Clostridium difficile-specific antibodies and uses thereof

ABSTRACT

The present invention is directed to  Clostridium difficile  toxin-specific antibodies, compositions, and uses thereof. The anti-toxin antibodies may be specific for either TcdA or TcdB. The invention also includes methods of treating a  Clostridium difficile  infection, methods of capturing  Clostridium difficile  toxins, and methods of detecting  Clostridium difficile  toxins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/881,398, filed Apr. 25, 2013, which is a national stageentry of international patent application number PCT/CA2011/001201,filed Oct. 25, 2011, and claims the benefit of U.S. provisional patentapplication Ser. No. 61/406,254, filed on Oct. 25, 2010, the entirecontents of all of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to Clostridium difficile-specificantibodies and uses thereof. More specifically, the present inventionrelates to Clostridium difficile toxin-specific antibodies and usesthereof.

BACKGROUND OF THE INVENTION

Clostridium difficile is a Gram-positive, anaerobic, endospore-forminggastrointestinal pathogen responsible for C. difficile-associateddisease (CDAD) in humans and animals with symptoms ranging in severityfrom mild cases of antibiotic-associated diarrhea to fatalpseudomembranous colitis (Rupnik et al, 2009; Leffler and Lamont, 2009;Songer, 2004; Kelly et al, 1994). Each year in North America, 1-3% ofhospitalized patients receiving antibiotics become infected with C.difficile, leading to thousands of deaths and over $1 billion inassociated costs to the health-care system (Wilkins and Lyerly, 2003;Kyne et al, 2002; Kelly et al, 1994). C. difficile produces two primaryvirulence factors, toxin A (TcdA) and toxin B (TcdB), which are large(308 kDa and 269 kDa, respectively), single-subunit exotoxins composedof a catalytic, a translocation and a cell-receptor binding domain (RBD)(Jank and Aktories, 2008; Jank et al, 2007). Recently it was suggestedTcdB is solely responsible for C. difficile virulence (Lyras et al,2009), although earlier studies have shown both anti-TcdA and anti-TcdBmonoclonal antibodies (mAbs) were required for full protection ofhamsters from CDAD (Babcock et al, 2006; Kink and Williams, 1998) andanti-TcdA mAbs were required for protection in mice (Corthier et al,1991).

The current approach for treating most CDAD infections involvesadministration of antibiotics, most commonly metronidazole or vancomycin(Leffler and Lamont, 2009). Antibiotic treatment places selectionpressure on the organism, can lead to antibiotic resistance, andsuppresses or eliminates beneficial commensal microbes. However, thereare several other emerging challenges warranting the development ofnovel therapeutics. First, there is no acute CDAD treatment targetingTcdA/B. These toxins are responsible for loss of epithelial barrierfunction in the colon by disrupting tight junctions and increasingmembrane permeability, causing diarrhea and promoting severeinflammation (Rupnik et al, 2009; Jank and Aktories, 2008). Second,hypervirulent strains of C. difficile, such as the NAP1/027 isolate,over-express TcdA and TcdB (Warny et al, 2005) and have been associatedwith increased mortality rates and disease severity (O'Connor et al,2009; Pépin et al, 2005). Third, an estimated 20-25% of patientssuffering from CDAD experience symptomatic relapse after the initialinfection is cleared, with 45% of these patients prone to subsequentrelapses (Johnson, 2009). Taken together, there is a need fornon-antibiotic based reagents which target and inhibit TcdA and TcdB forCDAD therapy.

Individuals who are asymptomatic C. difficile carriers and patients whoexperience mild cases of CDAD tend to possess high anti-toxin A titers(Kyne et al, 2001; Kyne et al, 2000; Warny et al, 1994; Viscidi et al,1983). Conversely, patients susceptible to relapsing C. difficileinfection have low anti-TcdA immunoglobulin titers, specifically IgM,IgG2 and IgG3 isotypes (Katchar et al, 2007; Kyne et al, 2001).TcdA-neutralizing secretory IgA antibodies are also thought to play arole in regulating CDAD severity (Johal et al 2004; Kelly et al 1992).Therefore, the introduction of anti-toxin antibodies to patientssuffering from severe C. difficile infection may be a therapeuticallyuseful approach.

A limited number of animal and human studies have illustrated theeffectiveness of anti-toxin Abs for treatment of CDAD. Babcock et al(2006) intravenously administered anti-TcdA and anti-TcdB mAbs tohamsters and found a significant reduction in hamster mortality inprophylactic, primary disease and relapse models when both anti-toxinmAbs were administered. A recently completed clinical trial involvingthese two humanized mAbs appears promising (Lowy et al, 2010). Inanother study, intravenous administration of anti-TcdA mAbs raisedagainst the RBD followed by oral challenge with C. difficile resulted inprotection of mice (Corthier et al, 1991). Elsewhere, a toxoid vaccinegiven by the intraperitoneal route to hamsters conferred protectionagainst oral C. difficile challenge (Giannasca et al, 1999) and micevaccinated with DNA encoding the TcdA RBD resulted in full protectionfrom oral TcdA challenge (Gardiner et al, 2009). In humans, a number ofuncontrolled studies have reported intravenous immunoglobulin (IVIG)therapy to be successful for the treatment of severe CDAD (Juang et al,2007; Hassoun and Ibrahim, 2007; McPherson et al, 2006; Wilcox, 2004;Salcedo et al, 1997; Leung et al, 1991). IVIG involves administration ofhigh concentrations (150-400 mg/kg) of human immunoglobulins fromhealthy donors which are thought to contain neutralizing anti-toxinantibodies as an estimated 60% of healthy adults have detectable TcdA-and TcdB-specific serum IgG antibodies (Viscidi et al, 1983).

Given that C. difficile toxins rely on attachment to epithelial cellsfor entry (Jank and Aktories, 2008; Jank et al, 2007), neutralizing thetoxins within the lower gastrointestinal tract with antibodies may blockthe first step in CDAD pathogenesis. In animals, orally administeredbovine immunoglobulin concentrate (BIC) containing TcdA and TcdBneutralizing IgGs were able to prevent hamster mortality when used as apropholyactic (Lyerly et al, 1991) and protected rats from theenterotoxic effects of TcdA in vivo (Kelly et al, 1996). Chicken IgYantibodies specific for toxin RBDs were shown to reduce hamstermortality when administered orally to infected animals (Kink andWilliams, 1998). In humans, there have been limited reports on CDADtherapy with orally delivered Abs. Tjellström et al (1993) reported thesuccessful treatment of a 3½ year old boy suffering from severe CDADwith IgA antibody orally. Warny et al (1999) and Kelly et al (1997)examined the passage of anti-toxin bovine IgG through the humangastrointestinal tract and found a significant reduction in IgGactivity, likely due to proteolytic degradation within the uppergastrointestinal tract. The limited success of both oral and systemicanti-toxin immunotherapy in clinical settings has likely been hamperedby the high immunoglobulin dose requirements (150-400 mg/kg), theassociated costs of these doses, and a lack of published clinical datashowing the effectiveness of these treatments.

Despite such advances, there remains a need in the art for a safe andeffective therapeutic for treating C. difficile-associated disease aswell as for sensitive and effective reagents for the detection of toxinsA and B, the factors responsible for C. difficile-associated disease.

SUMMARY OF THE INVENTION

The present invention relates to Clostridium difficile-specificantibodies and uses thereof. More specifically, the present inventionrelates to Clostridium difficile toxin-specific antibodies and usesthereof.

The present invention provides an isolated or purified antibody orfragment thereof, comprising

-   -   a sequence of complementarity determining region (CDR) 1        selected from GRTFNTLS (SEQ ID NO:1); GRTFSMYR (SEQ ID NO:2);        GRTLSSYI (SEQ ID NO:3); GRTFSMDP (SEQ ID NO:4); IRSFSNRN (SEQ ID        NO:5); and ERTFSRYP (SEQ ID NO:6);    -   a sequence of CDR2 selected from VSRSGGST (SEQ ID NO:7);        ITRNGSST (SEQ ID NO:8); ISRRGGNS (SEQ ID NO:9); GSSTGRTT (SEQ ID        NO:10); ISWGGGST (SEQ ID NO:11); and ISSTGTST (SEQ ID NO:12);        and    -   a sequence of CDR3 selected from AAAATKSNTTAYRLSFDY (SEQ ID        NO:13); AATSGSSYLDAAHVYDY (SEQ ID NO:14); AADGSVAGWGRRSVSVSSYDY        (SEQ ID NO:15); AAAPYGANWYRDEYAY (SEQ ID NO:16);        AAEFGHNIATSSDEYDY (SEQ ID NO:17); and AVNSQRTRLQDPNEYDY (SEQ ID        NO:18),

wherein the antibody or fragment thereof is specific for TcdA. Theisolated or purified antibody or fragment thereof as described above maybe selected from the group consisting of:

-   -   an isolated or purified antibody or fragment thereof comprising        a sequence of CDR 1 of GRTFNTLS (SEQ ID NO:1); CDR2 of VSRSGGST        (SEQ ID NO:7); and CDR3 of AAAATKSNTTAYRLSFDY (SEQ ID NO:13);    -   an isolated or purified antibody or fragment thereof comprising        a sequence of CDR 1 of GRTFSMYR (SEQ ID NO:2); CDR2 of ITRNGSST        (SEQ ID NO:8); and CDR3 of AATSGSSYLDAAHVYDY (SEQ ID NO:14);    -   an isolated or purified antibody or fragment thereof comprising        a sequence of CDR 1 of GRTLSSYI (SEQ ID NO:3); CDR2 of ISRRGGNS        (SEQ ID NO:9); and CDR3 of AADGSVAGWGRRSVSVSSYDY (SEQ ID NO:15);    -   an isolated or purified antibody or fragment thereof comprising        a sequence of CDR 1 of GRTFSMDP (SEQ ID NO:4); CDR2 of GSSTGRTT        (SEQ ID NO:10); and CDR3 of AAAPYGANWYRDEYAY (SEQ ID NO:16);    -   an isolated or purified antibody or fragment thereof comprising        a sequence of CDR 1 of IRSFSNRN (SEQ ID NO:5); CDR2 of ISWGGGST        (SEQ ID NO:11); and CDR3 of AAEFGHNIATSSDEYDY (SEQ ID NO:17); or    -   an isolated or purified antibody or fragment thereof comprising        a sequence of CDR 1 of ERTFSRYP (SEQ ID NO:6); CDR2 of ISSTGTST        (SEQ ID NO:12); and CDR3 of AVNSQRTRLQDPNEYDY (SEQ ID NO:18).

The isolated or purified antibody or fragment thereof described abovemay comprise a sequence selected from the group consisting of:

(SEQ ID NO: 34) QVKLEESGGGLVQAGGSLRLSCAASGRTFNTLSMGWFRQAPGKEREFVAAVSRSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAAATKSNTTAYRLSFDYWGQGTQVTVSS; (SEQ ID NO: 35)QVKLEESGGGLVQAGGSLRLSCAASGRTFSMYRMGWFRQAPGKEREFVGVITRNGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATSGSSYLDAAHVYDYWGQGTQVTVSS; (SEQ ID NO: 36)QVKLEESGGGLVQPGGSLRLSCAASGRTLSSYIVAWFRQAPGKEREFVAGISRRGGNSAYVESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADGSVAGWGRRSVSVSSYDYWGQGTQVTVSS; (SEQ ID NO: 37)QVQLVESGGGLAQAGGSLRLSCAASGRTFSMDPMAWFRQPPGKEREFVAAGSSTGRTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAAPYGANVVYRDEYAYWGQGTQVTVSS; (SEQ ID NO: 38)QVQLVESGGGLVQAGGSLRLSCAASIRSFSNRNMGWFRQPPGKEREFVAGISWGGGSTRYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEFGHNIATSSDEYDYWGQGTQVTVSS; (SEQ ID NO: 39)QVKLEESGGGLVQAGGSLRLSCAASERTFSRYPVAWFRQAPGAEREFVAVISSTGTSTYYADSVKGRFTISRDNAKVTVYLQMNNLKREDTAVYFCAVNSQRTRLQDPNEYDYWGQGTQVTVSS; (SEQ ID NO: 45)QVKLEESGGGLVQAGGSLRLSCAASGRTFNTLSMGWFRQAPGKEREFVCAVSRSGGSTYYADSVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVYYCAAAATKSNTTAYRLSFDYWGQGTQVTVSS; (SEQ ID NO: 46)QVKLEESGGGLVQAGGSLRLSCAASGRTFSMYRMGWFRQAPGKEREFVCVITRNGSSTYYADSVKGRFTCSRDNAKNTVYLQMNSLKPEDTALYYCAATSGSSYLDAAHVYDYWGQGTQVTVSS; (SEQ ID NO: 47)QVKLEESGGGLVQPGGSLRLSCAASGRTLSSYIVAWFRQAPGKEREFVCGISRRGGNSAYVESVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVYYCAADGSVAGWGRRSVSVSSYDYWGQGTQVTVSS; (SEQ ID NO: 48)QVQLVESGGGLAQAGGSLRLSCAASGRTFSMDPMAWFRQPPGKEREFVCAGSSTGRTTYYADSVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVYYCAAAPYGANWYRDEYAYWGQGTQVTVSS; (SEQ ID NO: 49)QVQLVESGGGLVQAGGSLRLSCAASIRSFSNRNMGWFRQPPGKEREFVCGISWGGGSTRYADSVKGRFTCSRDNAKKTVYLQMNSLKPEDTAVYYCAAEFGHNIATSSDEYDYWGQGTQVTVSS; and (SEQ ID NO: 50)QVKLEESGGGLVQAGGSLRLSCAASERTFSRYPVAWFRQAPGAEREFVCVISSTGTSTYYADSVKGRFTCSRDNAKVTVYLQMNNLKREDTAVYFCAVNSQRTRLQDPNEYDYWGQGTQVTVSS,or a sequence substantially identical thereto.

In another aspect, the present invention provides an isolated orpurified antibody or fragment thereof, comprising

-   -   a sequence of complementarity determining region (CDR) 1        selected from GNIFSINT (SEQ ID NO:19); GRTASGYG (SEQ ID NO:20);        GRTFSSGV (SEQ ID NO:21); GLSRYA (SEQ ID NO:22); and GSISRIST        (SEQ ID NO:23);    -   a sequence of CDR2 selected from ITSGGTT (SEQ ID NO:24);        ISRSGAGT (SEQ ID NO:25); ITTGGST (SEQ ID NO:26); TNWSSGNT (SEQ        ID NO:27); and ISTGGTT (SEQ ID NO:28); and    -   a sequence of CDR3 selected from NTVKVVGGRLDNPDY (SEQ ID NO:29);        VARPTKVDRDYATRREMYNY (SEQ ID NO:30); NSVAVVGGVIKSPDY (SEQ ID        NO:31); AARKLDVPSRYSQHYDY (SEQ ID NO:32); and AAGWKVVRGSLEYEY        (SEQ ID NO:33),        wherein the antibody or fragment thereof is specific for TcdB.        The isolated or purified antibody or fragment thereof as just        described may be selected from the group consisting of:    -   an isolated or purified antibody or fragment thereof comprising        a sequence of CDR 1 of GNIFSINT (SEQ ID NO:19); CDR2 of ITSGGTT        (SEQ ID NO:24); and CDR3 of NTVKVVGGRLDNPDY (SEQ ID NO:29);    -   an isolated or purified antibody or fragment thereof comprising        a sequence of CDR 1 of GRTASGYG (SEQ ID NO:20); CDR2 of ISRSGAGT        (SEQ ID NO:25); and CDR3 of VARPTKVDRDYATRREMYNY (SEQ ID NO:30);    -   an isolated or purified antibody or fragment thereof comprising        a sequence of CDR 1 of GRTFSSGV (SEQ ID NO:21); CDR2 of ITTGGST        (SEQ ID NO:26); and CDR3 of NSVAVVGGVIKSPDY (SEQ ID NO:31);    -   an isolated or purified antibody or fragment thereof comprising        a sequence of CDR 1 of GLSRYA (SEQ ID NO:22); CDR2 of TNWSSGNT        (SEQ ID NO:27); and CDR3 of AARKLDVPSRYSQHYDY (SEQ ID NO:32); or    -   an isolated or purified antibody or fragment thereof comprising        a sequence of CDR 1 of GSISRIST (SEQ ID NO:23); CDR2 of ISTGGTT        (SEQ ID NO:28); and CDR3 of AAGWKVVRGSLEYEY (SEQ ID NO:33).

The isolated or purified antibody or fragment thereof as described abovemay comprise a sequence selected from the group consisting of:

(SEQ ID NO: 40) QVQLVESGGGLVQPGGSLRLSCAASGNIFSINTMGWYRQAPGKQLELVAAITSGGTTSYTDSVEGRFTISRDNAKNAVYLQMNSLKAEDTAVYYCNTVKVVGGRLDNPDYWGQGTQVTVSS; (SEQ ID NO: 41)QVKLEESGGGLVQPGGSLRLSCAASGRTASGYGMGWFRQAPGKEREFVAAISRSGAGTLNADFVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVARPTKVDRDYATRREMYNYWGQGTQVTVSS; (SEQ ID NO: 42)QVKLEESGGGLVQAGGSLRLSCSASGRTFSSGVMGWFRQAPGKQRELVAAITTGGSTSYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNSVAVVGGVIKSPDYWGQGTQVTVSS; (SEQ ID NO: 43)QVQLVESGGGSVQAGGSLRLSCAASGLSRYAMAWFRQGTGKEREFVASTNWSSGNTPYADSVKGRFIISRDNAKNTVYLQMNSLKPGDTAIYYCAARKLDVPSRYSQHYDYWGQGTQVTVSS; (SEQ ID NO: 44)QVQLVESGGDLVQAGGSLRLSCAASGSISRISTMGWYRQAPGKQRELVATISTGGTTNYAESVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCAAGWKVVRGSLEYEYSGQGTQVTVSS; (SEQ ID NO: 51)QVQLVESGGGLVQPGGSLRLSCAASGNIFSINTMGWYRQAPGKQLELVCAITSGGTTSYTDSVEGRFTCSRDNAKNAVYLQMNSLKAEDTAVYYCNTVKVVGGRLDNPDYWGQGTQVTVSS, referred to herein as B5.2m;(SEQ ID NO: 52) QVKLEESGGGLVQPGGSLRLSCAASGRTASGYGMGWFRQAPGKEREFVCAISRSGAGTLNADFVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVYYCVARPTKVDRDYATRREMYNYWGQGTQVTVSS, referred to herein as B7.3m;(SEQ ID NO: 53) QVKLEESGGGLVQAGGSLRLSCSASGRTFSSGVMGWFRQAPGKQRELVCAITTGGSTSYTDSVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVYYCNSVAVVGGVIKSPDYWGQGTQVTVSS, referred to herein as B13.6m;(SEQ ID NO: 54) QVQLVESGGGSVQAGGSLRLSCAASGLSRYAMAWFRQGTGKEREFVCSTNWSSGNTPYADSVKGRFICSRDNAKNTVYLQMNSLKPGDTAIYYCAARKLDVPSRYSQHYDYWGQGTQVTVSS, referred to herein as B15.3m; and(SEQ ID NO: 55) QVQLVESGGDLVQAGGSLRLSCAASGSISRISTMGWYRQAPGKQRELCATISTGGTTNYAESVKGRFTCSRDNAKNTMYLQMNSLKPEDTAVYYCAAGWKVVRGSLEYEYSGQGTQVTVS, referred to herein as B15.5m,or a sequence substantially identical thereto.

The isolated or purified antibody or fragment thereof as described abovemay be a single-domain antibody (sdAb); the sdAb may be of camelidorigin.

The isolated or purified antibody or fragment thereof of as describedherein may be in a multivalent display format.

The isolated or purified antibody or fragment thereof as describedherein may be immobilized onto a surface.

The isolated or purified antibody or fragment thereof of the presentinvention may be linked to a cargo molecule; the cargo molecule may be adetectable agent, a therapeutic, a drug, a peptide, a protease, anenzyme, a carbohydrate moiety, or a cytotoxic agent; one or moreliposomes loaded with a detectable agent, a therapeutic, a drug, apeptide, an enzyme, or a cytotoxic agent; or one or more nanoparticle,nanowire, nanotube, or quantum dots.

The present invention further encompasses a nucleic acid moleculeencoding the isolated or purified antibody or fragment thereof asdescribed above. The present invention also includes a vector comprisingthe nucleic acid molecule just described.

Also provided is a composition comprising one or more than one isolatedor purified antibody or fragment thereof of the present invention and apharmaceutically-acceptable carrier, diluent, or excipient.

The present invention further provides a method of treating aClostridium difficile infection, comprising administering the isolatedor purified antibody or fragment thereof of the present invention or thecomposition of described above to a subject in need thereof.

In another aspect, there is provided a method of capturing Clostridiumdifficile toxins, comprising contacting a sample with one or more thanone isolated or purified antibody or fragment thereof of the presentinvention immobilized onto a surface, and allowing the toxin(s) to bindto the isolated or purified antibody or fragment thereof. The methodjust described may further comprise identifying the toxin by massspectrometric methods and/or eluting the bound toxin.

The present invention additionally provides a method of detectingClostridium difficile toxins, comprising contacting a sample with one ormore than one isolated or purified antibody or fragment thereof linkedto a cargo molecule, and detecting the bound antibody or fragmentthereof using a suitable imaging or detection technology. The cargomolecule may be a detectable agent.

The present invention provides isolated llama single-domain antibodies(V_(H)Hs) capable of binding, detecting, capturing, and/or neutralizingC. difficile TcdA and TcdB. Without wishing to be bound by theory,V_(H)Hs targeting the toxin's receptor binding domain (RBD) may blockthe toxin-receptor interaction, thereby preventing toxin entry into thehost cell; a critical initial step in the TcdA/B mechanism of action(Jank and Aktories, 2008). To do so, a hyperimmunized llama V_(H)H phagedisplay library was constructed and panned with recombinant RBDfragments. The isolated V_(H)Hs were then characterized for theirability to bind native toxins and recombinant RBD fragments and thenature and relative positioning of epitopes. In addition, the ability ofV_(H)Hs to neutralize toxins in an in vitro cell cytotoxicity assay wasassessed.

Several TcdA-specific V_(H)Hs capable of neutralizing TcdA in vitrothrough high-affinity interactions with TcdA-RBD were found. V_(H)Hs areextremely stable antigen-binding domains that are expressed athigh-yields in recombinant organisms and are capable of neutralizinginfectious disease-related targets (Wesolowski et al, 2009). Withrespect to CDAD therapy, V_(H)Hs could be administered systemically totarget TcdA and TcdB as they share high sequence homology with humanV_(H) domains, thus are well-tolerated in humans (Vu et al, 1997;www.Ablynx.com). Enhanced toxin neutralizing efficacy may be obtained byincreasing their blood circulation half lives, size and avidity usingvarious techniques, including chimeric formats of anti-TcdA V_(H)Hslinked to an Fc domain, generation of bi- or tri-specific antibodyfusions with two or three anti-TcdA V_(H)Hs recognizing unique epitopes,PEGylation, fusion to serum albumin, or fusion to serum albumin-specificantibody fragments. By targeting C. difficile virulence factors such asTcdA/B, selection pressure is removed from the organism, decreasing thechance of antibiotic resistance. A mutation in the RBD, which isconserved among C. difficile isolates including hypervirulent 027ribotype strains, is unlikely to benefit the organism and in the eventit does occur, the toxin may lose its ability to enter host cells. Assuch, anti-TcdA/B V_(H)Hs are logical agents to explore for CDADtherapy.

In order to improve the V_(H)Hs' biophysical properties, the C.difficile TcdA-specific V_(H)Hs were engineered to insert anon-canonical disulfide bond by introducing Ala/Gly⁵⁴→Cys⁵⁴ andIle⁷⁸→Cys⁷⁸ mutations, allowing for the formation of a second,non-native disulfide bond between FR2 and FR3 in the V_(H)H hydrophobiccore. Disulfide bond formation was confirmed using a combination ofproteolytic and chemical digestion coupled with MS² to preciselyidentify V_(H)H peptide fragments harboring the introduced disulfidebond. The mutant antibodies were compared to their wild-typecounterparts with respect to yield, solubility, affinity for TcdA,thermal unfolding at neutral and acidic pH and protease resistance.Mutant V_(H)Hs were found to be soluble, non-aggregating monomers,possessing similar affinity constants to that of WT V_(H)Hs. SPR bindingexperiments revealed most mutant V_(H)Hs possessed 1- to 5-fold weakeraffinity constants relative to wild-type, which is consistent with otherreports in the art.

CD spectroscopy was used to compare wild-type and mutant V_(H)Hsecondary structure, tertiary structure, thermal stability (T_(m) andT_(onset)), and thermal refolding efficiency (TRE). Comparisons ofV_(H)H secondary and tertiary structure with far-UV and near-UV CDspectroscopy strongly suggested structural differences between wild-typeand mutants, at both neutral and acidic pH. For all mutants, peakintensity and selective peak minima shifts were observed, although theoverall spectral profiles remained very similar in all wild type/mutantpairs. More specifically, mutants consistently showed rightward peakshifts in the peak range of 230 nm-235 nm (far-UV CD spectra) and around297 nm (near-UV CD spectra) compared to wild type V_(H)Hs. Such patternsmay be used as signatures that could be used to quickly identify V_(H)Hscontaining a properly formed non-canonical disulfide bond, as couldSDS-PAGE motility values since, compared to wild type V_(H)Hs, mutantsconsistently moved slower in SDS-PAGE gels. Thus, the far- and near-UVCD spectral data suggests the introduced disulfide bond changes thestructure of V_(H)Hs. This is consistent with the observed perturbationsin affinities and specificities and increased GI protease resistance ofthe mutant V_(H)Hs compared to the wild types.

CD spectroscopy thermal denaturation experiments were performed to showa profound and significant (p<0.05) increase in the T_(m)s andT_(onset)s of mutant V_(H)Hs at both neutral and acidic pH. Increases inmidpoint temperature of unfolding (T_(m)) ranging from ˜4 to ˜12° C.were observed for all mutants, at both neutral and acidic pH. Withoutwishing to be bound by theory, for the mutant V_(H)Hs with a higherthermostability gain, the non-canonical disulfide linkage may have beena better fit to overall fold. For example, A19.2m and A24.1m showed thelowest thermostability gains and this would explain why they weretransformed into non-specific binders upon mutation. For A4.2m on theother hand, the non-canonical disulfide linkage seems to be a naturalfit, as it increased its T_(m) the most (by almost 12° C.) andsignificantly improved GI protease resistance (with the highest increasein pepsin resistance), all without adversely affecting the K_(D).

Digestion of the V_(H)Hs with major gastrointestinal proteases atbiologically relevant concentrations revealed a significant (p<0.05)increase in pepsin resistance for all mutants; however, increases inresistance profiles to chymotrypsin and trypsin were not as universal.Each wild-type and mutant V_(H)H pair possessed an identical number oftheoretical protease cleavage sites (data not shown); thus it seems thatthe added disulfide bond leads to a more compact and thermodynamicallystable V_(H)H structure, preventing pepsin and chymotrypsin fromaccessing proteolytic cleavage sites. V_(H)H refolding was also examinedusing CD spectroscopy. While wild-type refolding was better than mutantV_(H)H refolding at neutral pH the reverse was true under stringentconditions (acidic pH). At acidic pH, 5 of 6 mutant V_(H)Hs possessedgreater refolding efficiency than wild-type after complete thermaldenaturation with the majority essentially showing reversible thermalunfolding.

The introduction of the Cys54/Cys78 disulfide linkage into V_(H)Hs ledto increases in both T_(m) and thermodynamic stability. Proteins withhigher T_(m) are also less likely to unfold [62]. These may be thereasons why the present mutants were more resistant to acid-inducedunfolding at 37° C., supported by the higher T_(onset)s and pepsinresistance of the mutant V_(H)Hs. This benefit is not realized formutants against trypsin, possibly because their cleavage sites are athydrophilic residues (Lys or Arg), which may be in more exposed regionsof the V_(H)H, possibly located in the CDR regions. Further, theseregions would not be protected by stabilizing the core of the structure.An increase in T_(onset) temperatures for mutants at the physiologicalcondition representative of the stomach (pH 2.0 and 37° C.) to valuessignificantly above 37° C. (T_(onset)s from 45° C.-53° C.) was observed.This indicates that the mutants should remain fully folded at 37° C. inthe stomach, hence resisting pepsin degradation (and denaturation) to ahigher extent than wild type V_(H)Hs, which is supported by the pepsindigestion experiments herein.

The toxin A neutralizing efficacy of the disulfide bond mutant V_(H)Hswas 3-4 fold weaker compared to the wild-type V_(H)Hs in toxin Aneutralization in cell-based assays, presumably a reflection in thereduced affinities of 3 of 4 V_(H)Hs for the toxin. Under stringentconditions in vivo, the lower affinity mutants may actually be moreefficacious than the higher affinity wild-type V_(H)Hs due to theirgreater stability.

It is presently shown that the introduction of a second disulfide bondinto the hydrophobic core of a panel of llama V_(H)Hs increased thermalstability and GI protease resistance; the approach is both effective andgeneral. While affinity, specificity, and expression yield may bereduced, the mutants comprising additional disulfide bond outperformedthe wild-type V_(H)Hs under more stringent physiological conditions;this far outweighs the reductions in affinity.

Protein-based oral therapeutics have several conceived advantages oversystemic administration: convenience, patience compliance, lower cost,pain-free administration, drug purity, flexibility in production source(i.e., bacterial, plant, etc.), and fewer concerns over immunogenicity.Despite the many advantages of orally administering proteintherapeutics, few successes have been realized due to the destabilizingenvironment of the GI tract. Of the major GI proteases, pepsin isconsidered the primary cause of antibody degradation and hence a majorobstacle facing orally-delivered antibody therapeutics. The introductionof an additional disulfide bond in the hydrophobic core of the anti-TcdAV_(H)Hs not only increased thermal stability at neutral pH, but alsorepresents a generic strategy to increase antibody stability at low pHand impart pepsin resistance which is desirable for protein-based oraltherapeutics.

Additional aspects and advantages of the present invention will beapparent in view of the following description. The detailed descriptionand examples, while indicating preferred embodiments of the invention,are given by way of illustration only, as various changes andmodifications within the scope of the invention will become apparent tothose skilled in the art in light of the teachings of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will now be described by wayof example, with reference to the appended drawings, wherein:

FIG. 1A-FIG. 1E relates to the isolation of anti-TcdA/B V_(H)Hs. FIG. 1Ais a schematic representation of native TcdA/B and recombinant fragmentsof the cell receptor-binding domain (TcdA-RBD-f1 and TcdB-RBD-f1) usedfor llama immunization and library panning. Numbers represent the aminoacid residues of each toxin, labelled from the N-termini (1) toC-termini (2710/2366 for TcdA and TcdB, respectively). Drawings are notto scale. GT=glucosyltransferase domain; CP=cysteine protease domain;HR=hydrophobic membrane insertion domain; RBD=cell-receptor bindingdomain. FIG. 1B is an SDS-PAGE profile of the purified C. difficiletoxins (3 μg per lane; from C. difficile strain 10463) used herein. Theupper arrow shows full-length TcdA (308 kDa) and TcdB (269 kDa). FIG. 10shows ELISA results demonstrating a total llama serum response for therecombinant RBD fragments. Serum was prepared from llama blood drawn 57days after the initial immunization. Immune (A): immune serum againstTcdA-RBD-f1; Immune (B): immune serum against TcdB-RBD-f1; Pre-Immune:pre-immune serum against TcdA-RBD-f1. FIG. 1D shows ELISA resultsdemonstrating the llama heavy-chain IgG (HCAb) G1 fraction response wasspecific for the recombinant RBD fragments. Serum was fractionated fromllama blood drawn 84 days after the initial immunization and the G1fraction shown did not recognize PEB3 or CPS, two unrelated antigens.FIG. 1E shows the SDS-PAGE profile of purified V_(H)Hs (2 μg per lane)isolated from the hyperimmunized llama phage display library. Molecularweight markers (M) are given in kDa. NR: non-reducing.

FIG. 2 is an amino acid sequence alignment of the anti-TcdA/B V_(H)Hsisolated. Framework regions, FRs, and complementarity-determiningregions, CDRs (shaded in grey), are grouped according to the IMGTnumbering system (http://imgt.cines.fr/). Hallmark positions, 42, 49, 50and 52, where V_(H)Hs can be distinguished from V_(H) based on aminoacid identity are illustrated with asterisks. V_(H)s generally have V,G, L and W at positions 42, 49, 50 and 52, respectively; in manyV_(H)Hs, the aforementioned residues are replaced with F/Y, E/Q/R, R andF/L, as shown.

FIG. 3A-FIG. 3C describes the construction, expression and functionalcharacterization of pentameric versions of monomeric anti-TcdA V_(H)Hs.FIG. 3A is a schematic diagram of a V_(H)H pentamer. V_(H)H monomersexpressed as fusions to the verotoxin B (VTB) subunit self-assembly intopentameric structures in E. coli. FIG. 3B shows an SDS-PAGE gelcontaining eluted IMAC fractions of purified V_(H)H pentamers A5.1p andA20.1p. Molecular weight markers (M) are given in kDa. NR: non-reducing.Arrows indicate the location of purified VTB-V_(H)H monomers. FIG. 3Cshows ELISA results comparing the functional activity of V_(H)H monomers(A5.1 and A20.1) and V_(H)H pentamers (A5.1p and A20.1p) to wells coatedwith TcdA. Equimolar concentrations of monomer/pentamer were used in theassay relative to the V_(H)H monomer. Monomer/pentamer binding wasdetected with rabbit anti-His₆ IgG-HRP and absorbances were read at 450nm.

FIG. 4A-FIG. 4D depicts the toxin binding characteristics of V_(H)Hs.ELISA demonstrating the anti-TcdA V_(H)Hs (FIG. 4A) and anti-TcdBV_(H)Hs (FIG. 4B) recognize native toxins and recombinant RBD fragments.Wells were coated with molar equivalent concentrations. FIG. 4C and FIG.4D shows binding of various concentrations of V_(H)Hs to immobilizedTcdA or TcdB, respectively.

FIG. 5 shows the gel filtration profiles of anti-TcdA V_(H)Hs obtainedfrom a Superdex™ 75 column. The single peak for all WT V_(H)Hs suggestsall are non-aggregating monomeric species.

FIG. 6A-FIG. 6I shows surface plasmon resonance analysis of anti-TcdA/BV_(H)Hs. Sensorgrams of TcdA-specific V_(H)Hs A4.2, A5.1, A19.2, A20.1,A24.1 and A26.8 binding to immobilized TcdA (FIG. 6A-FIG. 6F) andTcdB-RBD-f1 (FIG. 1A) binding to immobilized TcdB-specific V_(H)Hs B5.2,B13.6 and B15.5 (FIG. 6G-FIG. 6I) are shown. In experiments involvingTcdA-specific V_(H)Hs, TcdA was immobilized on CM5-dextran chips andmonomeric V_(H)Hs were passed over at concentration ranges noted on eachsensorgram, giving affinity constants ranging from 2 nM to 290 nM. Inexperiments involving TcdB-specific V_(H)Hs, antibodies were immobilizedon CM5-dextran chips and TcdB-RBD-f1 ranging in concentration from 2 μMto 200 nM was passed over, giving affinity constants ranging from 100 nMto 400 nM. Black lines represent raw data measurements and grey linesrepresent fitted curves. All data presented here showed acceptablefitting to a 1:1 binding model. Rate and affinity constants are shown inTable 1.

FIG. 7A-FIG. 7D shows a subset of TcdB-specific V_(H)Hs with complexbinding to recombinant TcdB-RBD. Surface plasmon resonance sensorgramsfor V_(H)Hs that showed binding to TcdB-RBD-f1, but whose data was nonanalyzable. The recombinant TcdB-RBD-f1 fragment (2 μM→200 nM) waspassed over immobilized V_(H)Hs (526-1209 RUs). (A) B7.3, (B) B13.2, (C)B13.3, and (D) B15.3.

FIG. 8A-FIG. 8F shows the in vitro neutralization of TcdA cytotoxicitywith anti-TcdA V_(H)Hs. Dose-response curves of TcdA (FIG. 8A) and TcdB(FIG. 8B) on monolayers of IMR-90 human lung fibroblast (HLF) cells. Thepercentage of cell rounding was scored from 0% to 100% of the cellsaffected. FIGS. 8C-8E show neutralization of TcdA-induced cell roundingwith V_(H)Hs at 24 h post addition of 100 ng/ml TcdA. The finalconcentration of V_(H)Hs in each assay well were 1000 nM (FIG. 8C), 10nM (FIG. 8D) and 0.1 nM (FIG. 8E) and V_(H)Hs were added as singles,pairs, or triplet combinations. White bars represent single V_(H)Hs orPBS control, grey bars represent paired combinations and black barsrepresent triplet combinations. Combinations of V_(H)Hs (i.e., pairs andtriples) increased toxin neutralizing efficacy. FIG. 8F showsrepresentative photographs of TcdA neutralization with 1000 nM V_(H)Hwere taken 24 h post toxin addition. The black bar represents 100 μm.

FIG. 9A-FIG. 9C shows results of epitope typing experiments. Anti-toxinV_(H)Hs recognize conformational (A4.2, A5.1, A20.1, A26.8) and linear(A19.2) epitopes on native C. difficile toxin and recombinant fragmentsof the cell receptor-binding domain. FIG. 9A shows ELISA results on TcdAtreated with various temperatures for 30 min before probing withV_(H)Hs. At treatment temperatures above the TcdA midpoint unfoldingtemperature (T_(m)), binding of 4 out of 5 TcdA-specific V_(H)Hs wasabolished. The dotted line represents the TcdA T_(m) of ˜55° C. FIG. 9Bshows Western blots (reducing/denaturing) probed with His-taggedanti-TcdA V_(H)Hs or control mouse anti-TcdA IgG (PCG-4). Binding wasdetected with nickel-AP or goat anti-mouse IgG-AP, respectively. Of theV_(H)Hs tested, only A19.2 recognized denatured TcdA and the secondaryconjugates did not bind denatured TcdA. FIG. 9C shows Western blots(native-PAGE) probed with anti-TcdA V_(H)Hs or control PCG4. V_(H)Hs andPCG4 bound TcdA. The goat anti-mouse IgG-AP conjugates stronglycross-reacted with TcdA in the absence of PCG4.

FIG. 10A-FIG. 10B shows results of BIACORE co-injection experiments,which were used to determine if pairs of V_(H)Hs could bind TcdAsimultaneously. The BIACORE sensorgrams in FIG. 10A and FIG. 10B of allof the possible paired combinations of A4.2, A5.1, A20.1 and A26.8, inboth orientations, are shown. Dashed lines represent injection of asingle V_(H)H followed by injection of buffer. Solid lines representco-injections of the first V_(H)H followed by injection of a secondV_(H)H. For all experiments, 80 μl of each V_(H)H at a concentration 20×its K_(D) was injected over 10,287 RUs of immobilized TcdA at 40 μl/min.In general, A4.2, A5.1 and A26.8 appeared to share an overlappingepitope as no significant increase in response was found upon injectionof the second species. Conversely, A20.1 appeared to bind a distinct,non-overlapping epitope.

FIG. 11 shows BIACORE results indicating that a subset of V_(H)Hs bindTcdA at overlapping epitopes. The three V_(H)Hs suspected of sharing anoverlapping epitope (FIG. 7) were injected alone (A4.2, A5.1, or A26.8)and as a triplet mixture (“Mix”) over immobilized TcdA. The BIACOREsensorgram illustrates similar R_(max) (˜160-200 RUs) values forindividual V_(H)Hs with no increase in response upon injection of themixed population, indicating these antibodies recognize an overlappingepitope on TcdA. If the mixture of V_(H)Hs were free to bind atnon-overlapping sites, one would expect an R_(max) value for themixtures to reach the sum of all individual R_(max) values (i.e., ˜540RUs). In all experiments, 80 μl of V_(H)Hs were injected at 40 μl/minand used at 20× their K_(D) concentrations.

FIG. 12A-FIG. 12D shows results of BIACORE analysis, which revealed thattwo trisaccharides, CD-grease (CD) and Le^(X)-AmHex (Le^(X)), known tointeract at the carbohydrate binding sites of TcdA-RBD, did not inhibitV_(H)H binding to immobilized TcdA. All four neutralizing V_(H)Hs weretested and one representative example is shown. FIG. 12A shows theresponses of A26.8 binding TcdA, CD-grease binding TcdA and co-injectionof A26.8 and CD-grease. FIG. 12B shows the responses of A26.8 bindingTcdA, Le^(X)-AmHex binding TcdA and co-injection of A26.8 andLe^(X)-AmHex. FIGS. 12C and 12D show subtraction of the responsegenerated from either trisaccharide binding to TcdA from co-injectionexperiments reveals a near identical response to that of A26.8 alone, anindication that V_(H)H binding to TcdA is not inhibited by thetrisaccharides. In all experiments, V_(H)Hs were used at their K_(D)concentrations and trisaccharides at their apparent K_(D) (2 mM).

FIG. 13A-FIG. 13B shows BIACORE results indicating V_(H)H binding toTcdA does not impair binding of the trisaccharide CD-grease (CD), whichis known to bind TcdA-RBD. FIG. 13A shows the injection of A20.1 (A,grey and dashed lines), followed by injection of A20.1 (B, dashed line)or A20.1+CD-grease (B, grey line), and finally injection of BIACOREbuffer (C, grey and dashed lines). FIG. 13B shows the injection of A26.8(A, grey and dashed lines), followed by injection of A26.8 (B, dashedline) or co-injection of A26.8+CD-grease (B, grey line), and finallyinjection of BIACORE buffer (C, grey and dashed lines). These resultssuggest V_(H)H binding is not at or in the carbohydrate binding site onTcdA-RBD.

FIG. 14 shows the alignment and comparison of wild-type and mutantV_(H)H amino acid sequences. Wild-type V_(H)H sequences are shown with asingle disulfide bond between Cys²³ and Cys¹⁰⁴. A second disulfide bondwas introduced through mutation of Ala⁵⁴/Gly⁵⁴ and Ile⁷⁸ to Cys⁵⁴ andCys⁷⁸ in framework region (FR) 2 and FR3, respectively. Disulfide bondsare shown as black lines. Bolded residues illustrate the disulfidebond-linked peptides identified by nanoRPLC-ESI-MS analysis on CNBr andtrypsin digested mutant V_(H)Hs. Amino acid numbering and CDRdesignation is based on the IMGT system.

FIG. 15A shows a non-reducing (NR) SDS-PAGE analysis and Western blot(WB) probed with an anti-His₆ IgG on IMAC-purified mutant V_(H)Hs. M:molecular weight marker in kDa. FIG. 15B is a representative SDS-PAGEanalysis showing mutant V_(H)Hs run slower than the correspondingwild-type V_(H)Hs under non-reducing conditions.

FIG. 16A-FIG. 16B confirms the disulfide bond formation between residuesCys⁵⁴ and Cys⁷⁸ by MS². FIG. 16A shows a SDS-PAGE gel under non-reducing(NR) conditions of V_(H)Hs (3 μg per lane), which illustratesnear-complete digestion with CNBr and trypsin. Untreated A5.1m was addedas a control (Ctl). M: molecular weight marker in kDa. FIG. 16B shows aMaxEnt 3 deconvoluted CID-MS² spectrum of the m/z 526.25 (3+) ion of thedisulfide-linked peptide EFVCVITR (P1)-FTCSR (P2), encompassing theCys⁵⁴-Cys⁷⁸ disulfide bond, from CNBr/trypsin digested A5.1m. Amino acidpositions are based on the IMGT numbering system.

FIG. 17A-FIG. 17B shows the characterization of mutant anti-TcdA V_(H)Hswhich possessed a second disulfide bond in the hydrophobic core thatwere introduced by mutation of two amino acids to cysteine. FIG. 17Ashows a comparison of the Size exclusion chromatography (SEC) analysisof wild-type (WT) anti-TcdA V_(H)Hs and mutant anti-TcdA V_(H)Hs(bottom) obtained from a Superdex™ 75 column. Similar size exclusionprofiles were obtained for mutant and wild-type, indicating the seconddisulfide bond does not promote the formation of interdomaindisulfide-bonds or multimeric mutant V_(H)Hs. The elution volumes(V_(e)s) of SEC molecular weight standards are shown with arrows and arealigned relative to the A4.2 and A4.2m chromatograms. a: ovalbumin(MW=43.0 kDa, V_(e)=8.90 ml); b: carbonic anhydrase (MW=30.0 kDa,V_(e)=9.71 ml); c: typsin inhibitor (MW=20.1 kDa, V_(e)=11.06 ml); d:α-lactalbumin (MW=14.4 kDa, V_(e)=11.97 ml); e: vitamin B (MW=1.3 kDa,V_(e)=18.7 ml). The equation of the line of a standard curve generatedfrom these standards was LOG₁₀MW=−0.1539V_(e)+2.9949 (r²=0.9995). Fromthis equation the V_(H)H apparent MWs ranged from 9.8-13.6 kDa,indicating monomeric V_(H)Hs. FIG. 17B shows surface plasmon resonance(SPR) sensorgrams for four mutant V_(H)Hs binding to immobilized TcdA.Grey lines represent raw data measurements and black lines representfitted curves. Kinetic and affinity constants are given in Table 4.Binding of A19.2m and A24.1m to TcdA was non-specific, and the kineticand affinity constants could not be determined. The binding shows mutantV_(H)Hs with a second disulfide bond retain high-affinity binding toTcdA.

FIG. 18 shows far-UV CD spectra of V_(H)Hs at neutral and acidic pH. CDscans (210 nm-260 nm) were performed at 25° C. on V_(H)Hs (50 μg/mL)equilibrated for 2 h in 10 mM phosphate buffer (pH 7.3) or 10 mMphosphate buffer+50 mM HCl (pH 2.0). The spectra represent the meanresidue ellipticity of 8 data accumulations collected from 2 independentexperiments. Black lines: wild-type V_(H)H; grey lines: mutant V_(H)H.

FIG. 19 shows near-UV CD analysis of V_(H)Hs at neutral and acidic pH.CD scans (250 nm-340 nm) were performed at 25° C. on V_(H)Hs (250 μg/mL)equilibrated for 2 h in 10 mM phosphate buffer (pH 7.3) or 10 mMphosphate buffer+50 mM HCl (pH 2.0). The spectra represent the meanresidue ellipticity from 8 data accumulations collected from 2independent experiments. Black lines: wild-type V_(H)H; grey lines:mutant V_(H)H.

FIG. 20A-FIG. 20B shows far-UV CD scans used to determine V_(H)Hrefolding efficiencies under neutral and acidic pH conditions. A5.1 isshown as a representative example. FIG. 20A shows CD spectra collectedon equilibrated V_(H)Hs (50 μg/ml) at 25° C. before heat treatment (scan1, solid lines), after exposure to 96° C. for 20 min (scan 2, dottedlines), and after cooling to 25° C. for 3 h (scan 3, dashed lines).Scans are an average of 4 data accumulations. FIG. 20B shows a summaryof thermal refolding efficiencies at pH 7.3 and pH 2.0, calculated usingEquation 2 and following the changes in ellipticity at 215 nm. Dotsrepresent the mean thermal refolding efficiency (TRE) of individualV_(H)Hs from two independent experiments with 4 data accumulations ineach experiment. Bars represent the mean TRE of each group of V_(H)Hs.

FIG. 21A-FIG. 21C shows results of circular dichroism analysis for thedetermination of WT and mutant V_(H)H melting temperatures at neutraland acidic pH. FIG. 21A shows unfolding transition curves for all six WTand mutant anti-TcdA V_(H)Hs at neutral pH (7.3) and acidic pH (2).V_(H)H thermal unfolding midpoint temperatures (T_(m)s) were determinedusing CD spectroscopy by following antibody unfolding (50 μg/mL) at 215nm in 10 mM phosphate buffer+/−50 mM HCl. T_(m) was determined for eachcurve by Boltzmann non-linear curve fitting analysis in GraphPad Prism.The goodness of curve fit (r²) ranged from 0.9901-0.9995. In the case ofV_(H)Hs with few lower baseline data points the T_(m) is a minimalestimate. FIG. 21B and FIG. 21C show a summary of the V_(H)H T_(m)s andT_(onset)s, respectively. Each dot represents individual V_(H)H and thebar represents the mean T_(m) or T_(onset) value in each group. P-valueswere determined using the unpaired two-tailed t-test The T_(m) valuesare summarized in Table 6.

FIG. 22A-FIG. 22E shows the resistance profiles of WT and mutant V_(H)Hsto the major gastrointestinal proteases pepsin, trypsin andchymotrypsin. FIG. 22A is a representative SDS-PAGE gel comparing theprofiles of WT and mutant A5.1 V_(H)H after no treatment or digestionwith various ratios of pepsin for 1 h at 37° C. V_(H)H epitope tags(“tag”), consisting of c-Myc and His₆, were preferentially cleaved byall proteases (confirmed by mass spectrometry analysis—data not shown).Densitometric analysis of SDS-PAGE gels allowed for the determination ofa percent of retained V_(H)H structure, which was denoted percentresistance. FIG. 22B-FIG. 22D summarizes the percent resistance of WTand mutant V_(H)Hs to pepsin, trypsin, and chymotrypsin after digestionfor 1 h at 37° C. using a protease concentration of 100 μg/ml (1:2.4ratio of protease:V_(H)H). Error bars represent the SEM obtained from 3independent digestions for each V_(H)H. FIG. 22E shows a summary of theV_(H)H resistance to each protease. Dots represent the mean (n=3)protease resistance profile of each V_(H)H relative to undigestedcontrols and the black bars represent the median resistance of eachgroup. P-values were determined using the unpaired two-tailedMann-Whitney U test. WT: wild-type V_(H)H; Mut: mutant V_(H)H; Chymo:chymotrypsin. The percent V_(H)H resistance to each protease is given inTable 8. In A, 1:240 and 1:24 ratios correspond to pepsin concentrationsof 1 μg/ml and 10 μg/ml, respectively, in reaction mixtures.

FIG. 23A-FIG. 23C shows the correlation between V_(H)H resistance topepsin and thermal stability at pH 2. FIG. 23A is a graph showing linearregression between V_(H)H pepsin resistance and V_(H)H T_(m) at pH 2.0.Boxes show the wild-type (WT) and mutant (Mut) V_(H)Hs, respectively.Linear regression analysis gave a correlation coefficient of r²=0.735and a significantly non-zero slope of the line (p=0.0004). FIG. 23B is agraph showing linear regression between wild-type V_(H)H pepsinresistance and wild-type V_(H)H T_(onset) at pH 2.0. The T_(onset) isdefined as the temperature at which 5% of the V_(H)H is unfolded. Linearregression analysis gave a correlation coefficient of r²=0.975 and asignificantly non-zero slope of the line (p=0.0002). FIG. 23C shows SPRanalysis (bottom) on mutant V_(H)Hs digested with pepsin (100 μg/ml, 1h, 37° C.). The pepsin-treated V_(H)Hs retained their ability to bindsurface-immobilized TcdA. SDS-PAGE gel (top) shows untreated (lanes 1,3, 5, 7) and pepsin-digested (lanes 2, 4, 6, 8) V_(H)Hs used for SPR.The contents of lanes 1 thru 8 are listed in the box. Normalizedk_(off)s for pepsin treated V_(H)Hs were similar to the k_(off) ofuntreated controls (box and Table 2). M: molecular weight markers inkDa; WT: wild-type V_(H)H; Mut: mutant V_(H)H; P: pepsin; R: reducingSDS-PAGE conditions.

FIG. 24 is a bar graph showing that mutant V_(H)Hs retainTcdA-neutralizing capacity. Confluent monolayers of IMR-90 human lungfibroblasts were incubated with TcdA or TcdA+V_(H)Hs for 24 h, and thepercentage of cells rounded was scored. V_(H)Hs (wild-type (WT) ormutant (Mut)) were added as pooled mixtures of A4.2, A5.1, A20.1, andA26.8 (250 nM each) or A4.2m, A5.1m, A20.1m, and A26.8m (250 nM each).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to Clostridium difficile-specificantibodies and uses thereof. More specifically, the present inventionrelates to Clostridium difficile toxin-specific antibodies and usesthereof.

The present invention provides isolated llama single-domain antibodies(V_(H)Hs) capable of binding and neutralizing C. difficile TcdA andTcdB. Without wishing to be bound by theory, V_(H)Hs targeting thetoxin's receptor binding domain (RBD) may block the toxin-receptorinteraction, thereby preventing toxin entry into the host cell; thisrepresents a critical initial step in the TcdA/B mechanism of action(Jank and Aktories, 2008).

Thus, the present invention provides an isolated or purified antibody orfragment thereof, comprising

-   -   a sequence of complementarity determining region (CDR) 1        selected from GRTFNTLS (SEQ ID NO:1); GRTFSMYR (SEQ ID NO:2);        GRTLSSYI (SEQ ID NO:3); GRTFSMDP (SEQ ID NO:4); IRSFSNRN (SEQ ID        NO:5); and ERTFSRYP (SEQ ID NO:6);    -   a sequence of CDR2 selected from VSRSGGST (SEQ ID NO:7);        ITRNGSST (SEQ ID NO:8); ISRRGGNS (SEQ ID NO:9); GSSTGRTT (SEQ ID        NO:10); ISWGGGST (SEQ ID NO:11); and ISSTGTST (SEQ ID NO:12);        and    -   a sequence of CDR3 selected from AAAATKSNTTAYRLSFDY (SEQ ID        NO:13); AATSGSSYLDAAHVYDY (SEQ ID NO:14); AADGSVAGWGRRSVSVSSYDY        (SEQ ID NO:15); AAAPYGANWYRDEYAY (SEQ ID NO:16);        AAEFGHNIATSSDEYDY (SEQ ID NO:17); and AVNSQRTRLQDPNEYDY (SEQ ID        NO:18),        wherein the antibody or fragment thereof is specific for TcdA.        The isolated or purified antibody or fragment thereof as just        described may comprise

a sequence of CDR 1 of GRTFNTLS (SEQ ID NO: 1);CDR2 of VSRSGGST (SEQ ID NO: 7); andCDR3 of AAAATKSNTTAYRLSFDY (SEQ ID NO: 13);a sequence of CDR 1 of GRTFSMYR (SEQ ID NO: 2);CDR2 of ITRNGSST (SEQ ID NO: 8); andCDR3 of AATSGSSYLDAAHVYDY (SEQ ID NO: 14);a sequence of CDR 1 of GRTLSSYI (SEQ ID NO: 3);CDR2 of ISRRGGNS (SEQ ID NO: 9); andCDR3 of AADGSVAGWGRRSVSVSSYDY (SEQ ID NO: 15);a sequence of CDR 1 of GRTFSMDP (SEQ ID NO: 4);CDR2 of GSSTGRTT (SEQ ID NO: 10); andCDR3 of AAAPYGANWYRDEYAY (SEQ ID NO: 16);a sequence of CDR 1 of IRSFSNRN (SEQ ID NO: 5);CDR2 of ISWGGGST (SEQ ID NO: 11); andCDR3 of AAEFGHNIATSSDEYDY (SEQ ID NO: 17); ora sequence of CDR 1 of ERTFSRYP (SEQ ID NO: 6);CDR2 of ISSTGTST (SEQ ID NO: 12); andCDR3 of AVNSQRTRLQDPNEYDY (SEQ ID NO: 18).

The present invention also provides an isolated or purified antibody orfragment thereof, comprising

-   -   a sequence of complementarity determining region (CDR) 1        selected from GNIFSINT (SEQ ID NO:19); GRTASGYG (SEQ ID NO:20);        GRTFSSGV (SEQ ID NO:21); GLSRYA (SEQ ID NO:22); and GSISRIST        (SEQ ID NO:23);    -   a sequence of CDR2 selected from ITSGGTT (SEQ ID NO:24);        ISRSGAGT (SEQ ID NO:25); ITTGGST (SEQ ID NO:26); TNWSSGNT (SEQ        ID NO:27); and ISTGGTT (SEQ ID NO:28); and    -   a sequence of CDR3 selected from NTVKVVGGRLDNPDY (SEQ ID NO:29);        VARPTKVDRDYATRREMYNY (SEQ ID NO:30); NSVAVVGGVIKSPDY (SEQ ID        NO:31); AARKLDVPSRYSQHYDY (SEQ ID NO:32); and AAGWKVVRGSLEYEY        (SEQ ID NO:33),        wherein the antibody or fragment thereof is specific for TcdB.        The isolated or purified antibody or fragment thereof as just        described may comprise

a sequence of CDR 1 of GNIFSINT (SEQ ID NO: 19);CDR2 of ITSGGTT (SEQ ID NO: 24); andCDR3 of NTVKVVGGRLDNPDY (SEQ ID NO: 29);a sequence of CDR 1 of GRTASGYG (SEQ ID NO: 20);CDR2 of ISRSGAGT (SEQ ID NO: 25); andCDR3 of VARPTKVDRDYATRREMYNY (SEQ ID NO: 30);a sequence of CDR 1 of GRTFSSGV (SEQ ID NO: 21);CDR2 of ITTGGST (SEQ ID NO: 26); andCDR3 of NSVAVVGGVIKSPDY (SEQ ID NO: 31);a sequence of CDR 1 of GLSRYA (SEQ ID NO: 22);CDR2 of TNWSSGNT (SEQ ID NO: 27); andCDR3 of AARKLDVPSRYSQHYDY (SEQ ID NO: 32); ora sequence of CDR 1 of GSISRIST (SEQ ID NO: 23);CDR2 of ISTGGTT (SEQ ID NO: 28); andCDR3 of AAGWKVVRGSLEYEY (SEQ ID NO: 33).

The term “antibody”, also referred to in the art as “immunoglobulin”(Ig), used herein refers to a protein constructed from paired heavy andlight polypeptide chains; various Ig isotypes exist, including IgA, IgD,IgE, IgG, and IgM. When an antibody is correctly folded, each chainfolds into a number of distinct globular domains joined by more linearpolypeptide sequences. For example, the immunoglobulin light chain foldsinto a variable (V_(L)) and a constant (C_(L)) domain, while the heavychain folds into a variable (V_(H)) and three constant (C_(H), C_(H2),C_(H3)) domains. Interaction of the heavy and light chain variabledomains (V_(H) and V_(L)) results in the formation of an antigen bindingregion (Fv). Each domain has a well-established structure familiar tothose of skill in the art.

The light and heavy chain variable regions are responsible for bindingthe target antigen and can therefore show significant sequence diversitybetween antibodies. The constant regions show less sequence diversity,and are responsible for binding a number of natural proteins to elicitimportant biochemical events. The variable region of an antibodycontains the antigen binding determinants of the molecule, and thusdetermines the specificity of an antibody for its target antigen. Themajority of sequence variability occurs in six hypervariable regions,three each per variable heavy (V_(H)) and light (V_(L)) chain; thehypervariable regions combine to form the antigen-binding site, andcontribute to binding and recognition of an antigenic determinant. Thespecificity and affinity of an antibody for its antigen is determined bythe structure of the hypervariable regions, as well as their size, shapeand chemistry of the surface they present to the antigen. Variousschemes exist for identification of the regions of hypervariability, thetwo most common being those of Kabat and of Chothia and Lesk. Kabat etal (1991) define the “complementarity-determining regions” (CDR) basedon sequence variability at the antigen-binding regions of the V_(H) andV_(L) domains. Chothia and Lesk (1987) define the “hypervariable loops”(H or L) based on the location of the structural loop regions in theV_(H) and V_(L) domains. As these individual schemes define CDR andhypervariable loop regions that are adjacent or overlapping, those ofskill in the antibody art often utilize the terms “CDR” and“hypervariable loop” interchangeably, and they may be so used herein.For this reason, the regions forming the antigen-binding site arepresently referred to herein as CDR L1, CDR L2, CDR L3, CDR H1, CDR H2,CDR H3 in the case of antibodies comprising a V_(H) and a V_(L) domain;or as CDR1, CDR2, CDR3 in the case of the antigen-binding regions ofeither a heavy chain or a light chain. The CDR/loops are referred toherein according to the IMGT numbering system (Lefranc, M.-P. et al.,2003), which was developed to facilitate comparison of variable domains.In this system, conserved amino acids (such as Cys23, Trp41, Cys104,Phe/Trp118, and a hydrophobic residue at position 89) always have thesame position. Additionally, a standardized delimitation of theframework regions (FR1: positions 1 to 26; FR2: 39 to 55; FR3: 66 to104; and FR4: 118 to 128) and of the CDR (CDR1: 27 to 38, CDR2: 56 to65; and CDR3: 105 to 117) is provided.

An “antibody fragment” as referred to herein may include any suitableantigen-binding antibody fragment known in the art. The antibodyfragment may be a naturally-occurring antibody fragment, or may beobtained by manipulation of a naturally-occurring antibody or by usingrecombinant methods. For example, an antibody fragment may include, butis not limited to a Fv, single-chain Fv (scFv; a molecule consisting ofV_(L) and V_(H) connected with a peptide linker), Fab, F(ab′)₂, singledomain antibody (sdAb; a fragment composed of a single V_(L) or V_(H)),and multivalent presentations of any of these.

In a non-limiting example, the antibody fragment may be an sdAb derivedfrom naturally-occurring sources. Heavy chain antibodies of camelidorigin (Hamers-Casterman et al, 1993) lack light chains and thus theirantigen binding sites consist of one domain, termed V_(H)H. sdAb havealso been observed in shark and are termed V_(NAR) (Nuttall et al,2003). Other sdAb may be engineered based on human Ig heavy and lightchain sequences (Jespers et al, 2004; To et al, 2005). As used herein,the term “sdAb” includes those sdAb directly isolated from V_(H),V_(H)H, V_(L), or V_(NAR) reservoir of any origin through phage displayor other technologies, sdAb derived from the aforementioned sdAb,recombinantly produced sdAb, as well as those sdAb generated throughfurther modification of such sdAb by humanization, affinity maturation,stabilization, solubilization, e.g., camelization, or other methods ofantibody engineering. Also encompassed by the present invention arehomologues, derivatives, or fragments that retain the antigen-bindingfunction and specificity of the sdAb.

A person of skill in the art would be well-acquainted with the structureof a single-domain antibody (see, for example, 3DWT, 2P42 in ProteinData Bank). An sdAb comprises a single immunoglobulin domain thatretains the immunoglobulin fold; most notably, only threeCDR/hypervariable loops form the antigen-binding site. However, and aswould be understood by those of skill in the art, not all CDR may berequired for binding the antigen. For example, and without wishing to belimiting, one, two, or three of the CDR may contribute to binding andrecognition of the antigen by the sdAb of the present invention. The CDRof the sdAb or variable domain are referred to herein as CDR1, CDR2, andCDR3, and numbered as defined by Lefranc, M.-P. et al. (2003).

As previously stated, the antibody or fragment thereof may be an sdAb.The sdAb may be of camelid origin or derived from a camelid V_(H)H, andthus may be based on camelid framework regions; alternatively, the CDRdescribed above may be grafted onto V_(NAR), V_(H)H, V_(H) or V_(L)framework regions. In yet another alternative, the hypervariable loopsdescribed above may be grafted onto the framework regions of other typesof antibody fragments (Fv, scFv, Fab). The present embodiment furtherencompasses an antibody fragment that is “humanized” using any suitablemethod know in the art, for example, but not limited to CDR grafting andveneering. Humanization of an antibody or antibody fragment comprisesreplacing an amino acid in the sequence with its human counterpart, asfound in the human consensus sequence, without loss of antigen-bindingability or specificity; this approach reduces immunogenicity of theantibody or fragment thereof when introduced into human subjects. In theprocess of CDR grafting, one or more than one of the heavy chain CDRdefined herein may be fused or grafted to a human variable region(V_(H), or V_(L)), or to other human antibody fragment framework regions(Fv, scFv, Fab). In such a case, the conformation of said one or morethan one hypervariable loop is preserved, and the affinity andspecificity of the sdAb for its target (i.e., toxins A and B) is alsopreserved. CDR grafting is known in the art and is described in at leastthe following: U.S. Pat. No. 6,180,370, U.S. Pat. No. 5,693,761, U.S.Pat. No. 6,054,297, U.S. Pat. No. 5,859,205, and European Patent No.626390. Veneering, also referred to in the art as “variable regionresurfacing”, involves humanizing solvent-exposed positions of theantibody or fragment; thus, buried non-humanized residues, which may beimportant for CDR conformation, are preserved while the potential forimmunological reaction against solvent-exposed regions is minimized.Veneering is known in the art and is described in at least thefollowing: U.S. Pat. No. 5,869,619, U.S. Pat. No. 5,766,886, U.S. Pat.No. 5,821,123, and European Patent No. 519596. Persons of skill in theart would also be amply familiar with methods of preparing suchhumanized antibody fragments and humanizing amino acid positions.

In a specific, non-limiting example, the antibody or fragment thereofthat is specific for TcdA may comprise a sequence selected from thegroup consisting of:

(SEQ ID NO: 34) QVKLEESGGGLVQAGGSLRLSCAASGRTFNTLSMGWFRQAPGKEREFVAAVSRSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAAATKSNTTAYRLSFDYWGQGTQVTVSS, referred to herein as A4.2;(SEQ ID NO: 35) QVKLEESGGGLVQAGGSLRLSCAASGRTFSMYRMGWFRQAPGKEREFVGVITRNGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATSGSSYLDAAHVYDYWGQGTQVTVSS, referred to herein as A5.1;(SEQ ID NO: 36) QVKLEESGGGLVQPGGSLRLSCAASGRTLSSYIVAWFRQAPGKEREFVAGISRRGGNSAYVESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADGSVAGWGRRSVSVSSYDYWGQGTQVTVSS, referred to herein as A19.2;(SEQ ID NO: 37) QVQLVESGGGLAQAGGSLRLSCAASGRTFSMDPMAWFRQPPGKEREFVAAGSSTGRTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAAPYGANWYRDEYAYWGQGTQVTVSS, referred to herein as A20.1;(SEQ ID NO: 38) QVQLVESGGGLVQAGGSLRLSCAASIRSFSNRNMGWFRQPPGKEREFVAGISWGGGSTRYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEFGHNIATSSDEYDYWGQGTQVTVSS, referred to herein as A24.1; and(SEQ ID NO: 39) QVKLEESGGGLVQAGGSLRLSCAASERTFSRYPVAWFRQAPGAEREFVAVISSTGTSTYYADSVKGRFTISRDNAKVTVYLQMNNLKREDTAVYFCAVNSQRTRLQDPNEYDYWGQGTQVTVSS, referred to herein as A 26.8,or a sequence substantially identical thereto.

In a specific, non-limiting example, the antibody or fragment thereofthat is specific for TcdB may comprise a sequence selected from thegroup consisting of:

(SEQ ID NO: 40) QVQLVESGGGLVQPGGSLRLSCAASGNIFSINTMGWYRQAPGKQLELVAAITSGGTTSYTDSVEGRFTISRDNAKNAVYLQMNSLKAEDTAVYYCNTVKVVGGRLDNPDYWGQGTQVTVSS, referred to herein as B5.2; (SEQ ID NO: 41)QVKLEESGGGLVQPGGSLRLSCAASGRTASGYGMGWFRQAPGKEREFVAAISRSGAGTLNADFVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVARPTKVDRDYATRREMYNYWGQGTQVTVSS, referred to herein as B7.3;(SEQ ID NO: 42) QVKLEESGGGLVQAGGSLRLSCSASGRTFSSGVMGWFRQAPGKQRELVAAITTGGSTSYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNSVAVVGGVIKSPDYWGQGTQVTVSS, referred to herein as B13.6;(SEQ ID NO: 43) QVQLVESGGGSVQAGGSLRLSCAASGLSRYAMAWFRQGTGKEREFVASTNWSSGNTPYADSVKGRFIISRDNAKNTVYLQMNSLKPGDTAIYYCAARKLDVPSRYSQHYDYWGQGTQVTVSS, referred to herein as B15.3; and(SEQ ID NO: 44) QVQLVESGGDLVQAGGSLRLSCAASGSISRISTMGWYRQAPGKQRELVATISTGGTTNYAESVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCAAGWKVVRGSLEYEYSGQGTQVTVSS, referred to herein as B15.5,or a sequence substantially identical thereto

A substantially identical sequence may comprise one or more conservativeamino acid mutations. It is known in the art that one or moreconservative amino acid mutations to a reference sequence may yield amutant peptide with no substantial change in physiological, chemical,physico-chemical or functional properties compared to the referencesequence; in such a case, the reference and mutant sequences would beconsidered “substantially identical” polypeptides. Conservative aminoacid mutation may include addition, deletion, or substitution of anamino acid; a conservative amino acid substitution is defined herein asthe substitution of an amino acid residue for another amino acid residuewith similar chemical properties (e.g. size, charge, or polarity).

In a non-limiting example, a conservative mutation may be an amino acidsubstitution. Such a conservative amino acid substitution may substitutea basic, neutral, hydrophobic, or acidic amino acid for another of thesame group. By the term “basic amino acid” it is meant hydrophilic aminoacids having a side chain pK value of greater than 7, which aretypically positively charged at physiological pH. Basic amino acidsinclude histidine (His or H), arginine (Arg or R), and lysine (Lys orK). By the term “neutral amino acid” (also “polar amino acid”), it ismeant hydrophilic amino acids having a side chain that is uncharged atphysiological pH, but which has at least one bond in which the pair ofelectrons shared in common by two atoms is held more closely by one ofthe atoms. Polar amino acids include serine (Ser or S), threonine (Thror T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N),and glutamine (Gln or Q). The term “hydrophobic amino acid” (also“non-polar amino acid”) is meant to include amino acids exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg (1984). Hydrophobic aminoacids include proline (Pro or P), isoleucine (Ile or I), phenylalanine(Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp orW), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G).“Acidic amino acid” refers to hydrophilic amino acids having a sidechain pK value of less than 7, which are typically negatively charged atphysiological pH. Acidic amino acids include glutamate (Glu or E), andaspartate (Asp or D).

Sequence identity is used to evaluate the similarity of two sequences;it is determined by calculating the percent of residues that are thesame when the two sequences are aligned for maximum correspondencebetween residue positions. Any known method may be used to calculatesequence identity; for example, computer software is available tocalculate sequence identity. Without wishing to be limiting, sequenceidentity can be calculated by software such as NCBI BLAST2 servicemaintained by the Swiss Institute of Bioinformatics (and as found athttp://ca.expasy.org/tools/blast/), BLAST-P, Blast-N, or FASTA-N, or anyother appropriate software that is known in the art.

The substantially identical sequences of the present invention may be atleast 65% identical; in another example, the substantially identicalsequences may be at least 65, 70, 85, 90, 95, 96, 97, 98, 99, or 100%identical, or any percentage therebetween, at the amino acid level tosequences described herein. Importantly, the substantially identicalsequences retain the activity and specificity of the reference sequence.In a non-limiting embodiment, the difference in sequence identity may bedue to conservative amino acid mutation(s). By way of example only, andwithout wishing to be limiting in any manner, the V_(H)Hs of the presentinvention have between about 66% and 82% sequence identity (see Tables 5and 6). In another non-limiting example, the present invention may bedirected to an antibody or fragment thereof comprising a sequence atleast 98% identical to that of the V_(H)Hs described herein.

A substantially identical sequence as defined by the present inventionalso includes a mutation to introduce an additional non-canonicaldisulfide bond. For example, and without wishing to be limiting, thenon-canonical disulfide bond may be introduced between framework region(FR) 2 and FR3. In a specific, non-limiting example, the mutation may beAla/Gly⁵⁴→Cys⁵⁴ and/or Val/Ile⁷⁸→Cys⁷⁸ mutation (based on IMGTnumbering). In a further specific example, the antibody or fragmentthereof that is specific for TcdA may comprise a sequence selected fromthe group consisting of:

(SEQ ID NO: 45)

(SEQ ID NO: 46)

(SEQ ID NO: 47)

(SEQ ID NO: 48)

(SEQ ID NO: 49)

(SEQ ID NO: 50)

(SEQ ID NO: 51)

(SEQ ID NO: 52)

(SEQ ID NO: 53)

(SEQ ID NO: 54)

(SEQ ID NO: 55)

or a sequence substantially identical thereto, with the proviso that thesubstantially identical sequence retains the non-canonical disulfidebond.

The isolated or purified antibody or fragment thereof of the presentinvention may bind to a conformational or linear epitope. Aconformational epitope is formed by amino acid residues that arediscontinuous in sequence, but proximal in the three-dimensionalstructure of the antigen. In contrast, a linear epitope (also referredto in the art as a “sequential epitope”) is recognized by its linearamino acid sequence, or primary structure. The conformational and linearepitopes of the antibodies or fragments thereof of the present inventionrecognize conformational and linear epitopes located in the region ofTcdA responsible for cell-receptor binding.

The antibody or fragment thereof of the present invention may alsocomprise additional sequences to aid in expression, detection orpurification of a recombinant antibody or fragment thereof. Any suchsequences or tags known to those of skill in the art may be used. Forexample, and without wishing to be limiting, the antibody or fragmentthereof may comprise a targeting or signal sequence (for example, butnot limited to ompA), a detection/purification tag (for example, but notlimited to c-Myc or a His₅ or His₆), or a combination thereof. Inanother example, the additional sequence may be a biotin recognitionsite such as that described by Cronan et al in WO 95/04069 or Voges etal in WO/2004/076670. As is also known to those of skill in the art,linker sequences may be used in conjunction with the additionalsequences or tags, or may serve as a detection/purification tag.

The antibody or fragment thereof of the present invention may also be ina multivalent display format, also referred to herein as multivalentpresentation. Multimerization may be achieved by any suitable method ofknow in the art. For example, and without wishing to be limiting in anymanner, multimerization may be achieved using self-assembly molecules asdescribed in Zhang et al (2004a; 2004b) and WO2003/046560. The describedmethod produces pentabodies by expressing a fusion protein comprisingthe antibody or fragment thereof of the present invention and thepentamerization domain of the B-subunit of an AB₅ toxin family (Merritt& Hol, 1995); the pentamerization domain assembles into a pentamer,through which a multivalent display of the antibody or fragment thereofis formed. Each subunit of the pentamer may be the same or different,and may have the same or different specificity. Additionally, thepentamerization domain may be linked to the antibody or antibodyfragment using a linker; such a linker should be of sufficient lengthand appropriate composition to provide flexible attachment of the twomolecules, but should not hamper the antigen-binding properties of theantibody.

Other forms of multivalent display are also encompassed by the presentinvention. For example, and without wishing to be limiting, the antibodyor fragment thereof may be presented as a dimer, a trimer, or any othersuitable oligomer. This may be achieved by methods known in the art, forexample direct linking connection (Nielson et al, 2000), c-jun/Fosinteraction (de Kruif & Logtenberg, 1996), “Knob into holes” interaction(Ridgway et al, 1996). Another method known in the art formultimerization is to dimerize the antibody or fragment thereof using anFc domain, e.g., human Fc domains. The Fc domains may be selected fromvarious classes including, but not limited to, IgG, IgM, or varioussubclasses including, but not limited to IgG1, IgG2, etc. In thisapproach, the Fc gene in inserted into a vector along with the sdAb geneto generate a sdAb-Fc fusion protein (Bell et al, 2010; Iqbal et al,2010); the fusion protein is recombinantly expressed then purified. Forexample, and without wishing to be limiting in any manner, multivalentdisplay formats may encompass chimeric formats of anti-TcdA V_(H)Hslinked to an Fc domain, or bi- or tri-specific antibody fusions with twoor three anti-TcdA V_(H)Hs recognizing unique epitopes. Enhanced toxinneutralizing efficacy may also be obtained using various techniques,including PEGylation, fusion to serum albumin, or fusion to serumalbumin-specific antibody fragments; these approaches increase theirblood circulation half lives, size and avidity.

The present invention also encompasses nucleic acid sequences encodingthe molecules as described herein. The nucleic acid sequence may becodon-optimized for expression in various micro-organisms. The presentinvention also encompasses vectors comprising the nucleic acids as justdescribed. Furthermore, the invention encompasses cells comprising thenucleic acid and/or vector as described.

The present invention further encompasses the isolated or purifiedantibody or fragments thereof immobilized onto a surface using variousmethodologies; for example, and without wishing to be limiting, theantibody or fragment may be linked or coupled to the surface via His-tagcoupling, biotin binding, covalent binding, adsorption, and the like.Immobilization of the antibody or fragment thereof of the presentinvention may be useful in various applications for capturing, purifyingor isolating proteins. The solid surface may be any suitable surface,for example, but not limited to the well surface of a microtiter plate,channels of surface plasmon resonance (SPR) sensorchips, membranes,beads (such as magnetic-based or sepharose-based beads or otherchromatography resin), glass, plastic, stainless steel, a film, or anyother useful surface such as nanoparticles, nanowires and cantileversurfaces.

Thus, the present invention also provides a method of capturingClostridium difficile toxins, comprising contacting a sample (such as,but not limited to C. difficile culture supernatant, human/animalintestinal/colonic fluid, or any other suitable sample) with one or morethan one isolated or purified antibody or fragment thereof of thepresent invention. The isolated or purified antibody or fragmentsthereof may be immobilized onto a surface. The toxin(s) then bind to theisolated or purified antibody or fragment thereof and are thus captured.The toxins may then optionally be identified by mass spectrometricmethods and/or released or eluted from their interaction with theantibody or fragment thereof; methods for releasing or eluting boundmolecules are well-known to those of skill in the art (for example butnot limited to heat elution steps), as are spectrometric methods capableof detecting and identifying the toxin. The isolated or purifiedantibody or fragment thereof of the present invention provideparticularly robust affinity purification reagents due to theirresistance to acidic and heat elution steps.

The invention also encompasses the antibody or fragment thereof asdescribed above linked to a cargo molecule. The cargo molecule may beany suitable molecule. For example, and without wishing to be limitingin any manner, the cargo molecule may be a detectable agent, atherapeutic agent, a drug, a peptide, an enzyme, a protease, acarbohydrate moiety, a cytotoxic agent, one or more liposomes loadedwith any of the previously recited types of cargo molecules, or one ormore nanoparticle, nanowire, nanotube, or quantum dots. For example, andwithout wishing to be limiting in any manner, the cargo molecule may bea protease that may digest the C. difficile toxin; in a furthernon-limiting example, the protease may be linked to a V_(H)H such as amutant V_(H)H that is protease resistant. In yet another non-limitingexample, the cargo molecule may be a cytotoxic agent that may beantibacterial or toxic towards host cells “infected” with C. difficiletoxins. In a further non-limiting example, the cargo molecule is aliposome, which makes the construct well-suited as a delivery agent formucosal vaccines. The cargo molecule may be linked to the antibody orfragment thereof by any suitable method known in the art. For example,and without wishing to be limiting, the cargo molecule may be linked tothe peptide by a covalent bond or ionic interaction. The linkage may beachieved through a chemical cross-linking reaction, or through fusionusing recombinant DNA methodology combined with any peptide expressionsystem, such as bacteria, yeast or mammalian cell-based systems. Methodsfor linking an antibody or fragment thereof to a therapeutic agent ordetectable agent would be well-known to a person of skill in the art.

The present invention also encompasses an antibody or fragment thereoflinked to a detectable agent. For example, the TcdA- or TcdB-specificantibody or fragment thereof may be linked to a radioisotope, aparamagnetic label, a fluorophore, an affinity label (for examplebiotin, avidin, etc), fused to a detectable protein-based molecule,nucleotide, quantum dot, nanoparticle, nanowire, or nanotube or anyother suitable agent that may be detected by imaging methods. In aspecific, non-limiting example, the antibody or fragment thereof may belinked to a fluorescent agent such as FITC or may genetically be fusedto the Enhanced Green Fluorescent Protein (EGFP). The antibody orfragment thereof may be linked to the detectable agent using any methodknown in the art (recombinant technology, chemical conjugation, etc.).

Thus, the present invention further provides a method of detectingClostridium difficile toxins, comprising contacting a sample (such as,but not limited to C. difficile culture supernatant, human/animalintestinal/colonic fluid, or any other suitable sample) with one or morethan one isolated or purified antibody or fragment thereof of thepresent invention. The isolated or purified antibody or fragmentsthereof may be linked to a detectable agent. The toxin(s) can then bedetected using detection and/or imaging technologies known in the art,such as, but not limited to mass spectrometric or immunoassay methods.

For example, and without wishing to be limiting in any manner, theisolated or purified antibody or fragment thereof linked to a detectableagent may be used in immunoassays (IA) including, but not limited toenzyme IA (EIA), ELISA, “rapid antigen capture”, “rapid chromatographicIA”, and “rapid EIA”. (For example, see Planche et al, 2008; Sloan etal, 2008; Rüssmann et al, 2007; Musher et al, 2007; Turgeon et al, 2003;Fenner et al, 2008)

The present invention also encompasses the isolated or purified antibodyor fragment thereof for detection of toxins in neutralized cell toxicityassays; methods for cell toxicity assays, also referred to herein ascytotoxicity assays, are known in the art and include, but are notlimited to those described by Planche et al (2008); Musher et al (2007);Turgeon et al (2003); and Fenner et al (2008). Cell cytotoxicity assaysinvolve incubating samples (for example, but not limited to patientstool samples) with cultured cells (for example, but not limited tofibroblasts) alone, or with the addition of a neutralizing agent, inthis case, the isolated or purified antibody or fragment thereof asdescribed herein. If the presence of the neutralizing agent reduces oreliminates cell toxicity observed with the cultured cells alone,presence of the toxins in the sample is confirmed. This type of assay isthe practical gold standard for CDAD detection in hospital diagnosticlaboratories.

The present invention also encompasses a composition comprising one ormore than one isolated or purified antibody or fragment thereof asdescribed herein. The composition may comprise a single antibody orfragment as described above, or may be a mixture of antibodies orfragments. Furthermore, in a composition comprising a mixture ofantibodies or fragments of the present invention, the antibodies mayhave the same specificity, or may differ in their specificities; forexample, and without wishing to be limiting in any manner, theantibodies or fragments may be specific to TcdA or TcdB, or a portion ofthe antibodies may be specific to TcdA while the other portion isspecific to TcdB.

The composition may also comprise a pharmaceutically acceptable diluent,excipient, or carrier. The diluent, excipient, or carrier may be anysuitable diluent, excipient, or carrier known in the art, and must becompatible with other ingredients in the composition, with the method ofdelivery of the composition, and is not deleterious to the recipient ofthe composition. The composition may be in any suitable form; forexample, the composition may be provided in suspension form, powder form(for example, but limited to lyophilised or encapsulated), capsule ortablet form. For example, and without wishing to be limiting, when thecomposition is provided in suspension form, the carrier may comprisewater, saline, a suitable buffer, or additives to improve solubilityand/or stability; reconstitution to produce the suspension is effectedin a buffer at a suitable pH to ensure the viability of the antibody orfragment thereof. Dry powders may also include additives to improvestability and/or carriers to increase bulk/volume; for example, andwithout wishing to be limiting, the dry powder composition may comprisesucrose or trehalose. In a specific, non-limiting example, thecomposition may be so formulated as to deliver the antibody or fragmentthereof to the gastrointestinal tract of the subject. Thus, thecomposition may comprise encapsulation, time-release, or other suitabletechnologies for delivery of the antibody or fragment thereof. It wouldbe within the competency of a person of skill in the art to preparesuitable compositions comprising the present compounds.

The present invention also comprises a method of treating a Clostridiumdifficile infection, comprising administering the isolated or purifiedantibody or fragment thereof of the present invention, or a compositioncomprising the antibody or fragment thereof, to a subject in needthereof. Any suitable method of delivery may be used. For example, andwithout wishing to be limiting in any manner, the antibody or fragmentthereof, or the composition, may be delivered systemically (orally,nasally, intravenously, etc.) or may be delivered to thegastrointestinal tract. Those of skill in the art would be familiar withsuch methods of delivery.

The present invention will be further illustrated in the followingexamples. However, it is to be understood that these examples are forillustrative purposes only and should not be used to limit the scope ofthe present invention in any manner.

Example 1: Purification of Toxins and Recombinant Fragments

C. difficile-associated diseases (CDAD) are caused by two high-molecularweight toxins composed of enzymatic, translocation, and cell-receptorbinding domains (RBD; FIG. 1A). TcdA and TcdB toxins were purified fromnatural sources, and recombinant fragments thereof were prepared.

TcdA and TcdB were isolated from Clostridium difficile strain 10463(ATCC, Manassas, Va.) as described previously (Keel and Songer, 2007)and were stored in 50 mM Tris-HCl buffer pH 7.5 at 4° C. The SDS-PAGEprofile of the purified C. difficile toxins (3 μg per lane; from strain10463) used is shown in FIG. 1B.

Recombinant fragments of TcdA (amino acid residues 2304-2710) and TcdB(amino acid residues 2286-2366), which are fragments of the RBD, werecloned (as a BamHI-HindIII fragment for tcdA and a BamHI-EcoRI fragmentfor tcdB) into pTrcHisB (Invitrogen, Carlsbad, Calif.), transforming E.coli DH5αMCR. Expression was induced by IPTG, cells harvested and lysedin a French pressure cell, and proteins TcdA-RBD-f1 and TcdB-RBD-f1purified by immobilized metal-affinity chromatography (IMAC).Recombinant RBD fragments were dialyzed into phosphate-buffered saline(PBS) pH 7.3 and stored at 4° C.

Example 2: Llama Immunization and Serum Response

To isolate V_(H)Hs which target the RBD of toxin A (TcdA) and toxin B(TcdB), a llama was immunized with recombinant RBD fragments TcdA-RBD-f1and TcdB-RBD-f1 (FIG. 1A) obtained in Example 1.

One male llama (Lama glama) was immunized by sub-cutaneous, lower-backinjection of TcdA-RBD-f1 and TcdB-RBD-f1 antigens. On Day 1, 200 μg ofeach antigen diluted in PBS to 1 ml was injected with 1 ml of Freund'sComplete Adjuvant (Sigma, St. Louis, Mo.). Three more injections of 100μg of each antigen+Freund's Incomplete Adjuvant (Sigma) were performedon Days 22, 36, and 50. A final injection of 100 μg of each antigen withno adjuvant was performed on Day 77. Pre-immune blood was drawn beforethe first injection on Day 1 and served as a negative control. Blood(10-15 ml) was collected on Days 29, 43, 57 and 84. Pre-immune andpost-immune total serum was analyzed for a specific response toTcdA-RBD-f1 and TcdB-RBD-f1 by ELISA on Day 57 (see below). Llama serafrom Day 84 were fractionated as previously described (Doyle et al,2008). The resulting fractions, A1 (HCAb), A2 (HCAb), G1 (HCAb) and G2(cIgG) were analyzed for specific binding to TcdA-RBD-f1 and TcdB-RBD-f1by ELISA. Briefly, 5 μg of TcdA-RBD-f1 or TcdB-RBD-f1 diluted in PBS wascoated overnight (100 μl/well, 18 h, 4° C.) in 96 well MAXISORP plates(Nalge Nunc International, Rochester, N.Y.). Plates were blocked withbovine serum albumin (BSA), washed with PBS-T (PBS+0.05% (v/v)Tween-20), and serial dilutions of pre-immune total serum, post-immunetotal serum (Day 57) and fractionated serum (Day 84) applied. Afterincubation at room temperature for 1.5 h and washing with PBS-T, goatanti-llama IgG (1:1,000 in PBS) was added for 1 h at 37° C. Afterwashing with PBS-T, pig anti-goat IgG-HRP conjugate (1:3,000 in PBS) wasadded for 1 h at 37° C. A final PBS-T wash precluded the addition of 100μl/well TMB substrate (KPL, Gaithersburg, Md.) for 10 min. The reactionwas stopped with 100 μl/well 1 M H₃PO₄ and read on a BioRad plate reader(Hercules, Calif.) at 450 nm.

An ELISA conducted on total serum from Day 57 clearly showed a specificimmune response for TcdA-RBD-f1 and TcdB-RBD-f1 compared to pre-immunesera collected before immunization on Day 0 (FIG. 1C). A second ELISAperformed on fractionated sera from Day 84 indicated the heavy-chain IgG(HCAb) and conventional IgG (cIgG) serum fractions recognizedTcdA-RBD-f1 and TcdB-RBD-f1. For example, the G1 HCAb fraction was shownto specifically recognize both recombinant fragments and did not bind totwo unrelated proteins PEB3 or CPS (FIG. 1D).

Example 3: Library Construction and Selection of Toxin-Binding V_(H)Hs

A hyperimmunized llama V_(H)H library was constructed based on RNAisolated from the serum collected in Example 2.

Library construction and panning was performed essentially as previouslydescribed (Arbabi-Ghahroudi et al, 2009c, 2009b; Tanha et al, 2003).Total RNA was isolated from approximately 5×10⁶ lymphocytes collected onday 84 post-immunization using the QIAAMP RNA Blood Mini Kit (QIAGEN,Mississauga, ON, Canada). About 5 μg of total RNA was used as templatefor first strand cDNA synthesis with oligo dT primers using theFirst-Strand cDNA Synthesis Kit (GE Healthcare, Baie-d'Urfé, QC,Canada). The cDNA was amplified by an equimolar mix of three variableregion-specific sense primers:

MJ1: (SEQ ID NO: 56) 5′-GCCCAGCCGGCCATGGCCSMKGTGCAGCTGGTGGAKTCTGGGGGA-3′ MJ2: (SEQ ID NO: 57)5′-CAGCCGGCCATGGCCCAGGTAAAGCTGGAGGAGTCTGG GGGA-3′ MJ3: (SEQ ID NO: 58)5′-GCCCAGCCGGCCATGGCCCAGGCTCAGGTACAGCTGGT GGAGTCT-3′,and two antisense CH₂-specific primers:

CH₂: (SEQ ID NO: 59) 5′-CGCCATCAAGGTACCAGTTGA-3′ CH₂b₃: (SEQ ID NO: 60)5′-GGTACCTGTCATCCACGGACCAGCTGA-3′.

Briefly, the PCR reaction mixture was set up in a total volume of 50 μlwith the following components: 1-3 μl cDNA, 5 pmol of MJ1-3 primermixture, 5 pmol of CH₂ or CH₂b₃ primers, 5 μl of 10× reaction buffer, 1μl of 10 mM dNTP, 2.5 unit of Taq DNA polymerase (Hoffmann-La Roche,Mississauga, ON, Canada). The PCR protocol consisted of an (i) initialstep at 94° C. for 3 min, (ii) followed by 30 cycles of 94° C. for 1min, 55° C. for 30 s, 72° C. for 30 s and (iii) a final extension stepat 72° C. for 7 min. The amplified PCR products were run in a 2% agarosegel and two major bands were observed: a band of about 850 bp,corresponding to conventional IgG, and a second band of around 600 bp,corresponding to heavy chain antibodies. The smaller bands were cut andpurified using the QIAQUICK Gel Extraction Kit (QIAGEN) and re-amplifiedin a second PCR in a total volume of 50 μl using 1 μl of DNA template, 5pmol of each of MJ7 primer (5′-CATGTGTAGACTCGCG GCCCAGCCGGCCATGGCC-3′SEQ ID NO:61) and MJ8 primer(5′-CATGTGTAGATTCCTGGCCGGCCTGGCCTGAGGAGACGGTGACCTGG-3′ SEQ ID NO:62), 5μl of 10× reaction buffer, 1 μl of 10 mM dNTP, 2.5 unit of Taq DNApolymerase. The PCR protocol consisted of (i) an initial step at 94° C.for 3 min, (ii) followed by 30 cycles of 94° C. for 30 s, 57° C. for 30s and 72° C. for 1 min and (iii) a final extension step at 72° C. for 7min. The amplified PCR products, ranging between 340 bp and 420 bp andcorresponding to V_(H)H fragments of heavy chain antibodies, werepurified using the QIAQUICK PCR Purification Kit (QIAGEN), digested withSfiI restriction enzyme (New England BioLabs, Pickering, ON, Canada) andre-purified using the same kit.

Eighty micrograms of pMED1 phagemid (Arbabi-Ghahroudi et al, 2009c) wasdigested with SfiI overnight at 50° C. To minimize self-ligation, 20units of XhoI and PstI restriction enzymes were added and the digestionreaction was incubated for an additional 2 h at 37° C. Sixty microgramsof digested phagemid DNA was ligated with 6 μg of digested V_(H)Hfragments for 3 h at room temperature using LigaFast Rapid DNA LigationSystem (Promega, Madison, Wis.) and its protocol. The ligated materialswere purified using the QIAQUICK PCR Purification Kit in a final volumeof 100 μl and electroporated in 5 μl portions into commercialelectrocompetent TG1 E. coli cells (Stratagene, La Jolla, Calif.) asdescribed (Arbabi-Ghahroudi et al, 2009c). The size of the library wasdetermined (Arbabi-Ghahroudi et al, 2009c) to be 5×10⁷. Colony-PCR andsequencing involving 20 colonies showed all tested clones had uniqueV_(H)Hs (Arbabi-Ghahroudi et al, 2009c). The library was grown for 2 hat 37° C., 250 rpm in the presence of 2% (w/v) glucose. The bacterialcells were pelleted, resuspended in 2×YT/Amp/Glu (2×YT medium with 100μg/ml ampicillin and 2% (w/v) glucose) with 35% (v/v) glycerol andstored at −80° C. in small aliquots.

Panning experiments were essentially performed as described (Arbabi etal, 1997). Five milliliters of the library (1.5×10⁸ cells) was thawed onice and grown in 2×YT/Amp/Glu for about 2 h at 37° C. (A₆₀₀=0.4-0.5).Cells were subsequently infected with 20× excess M13KO7 helper phage(New England Biolabs) for 1 h at 37° C. The culture was then centrifugedat 4° C. and infected cell pellets were resuspended in 200 ml of2×YT/Amp with 50 μg/ml kanamycin and incubated at 37° C. and 250 rpm.The phage particles in culture supernatant were incubated with ⅕ volumeof 20% PEG 6000/2.5M NaCl on ice for 1 h and centrifuged at 10,000 rpmfor 15 min. The phage pellets were resuspended in 1.5 ml of sterile PBS,titrated and used as input phage for panning. For panning, 96-wellMAXISORP plates were coated with 20 μg of TcdA-RBD-f1 or TcdB-RBD-f1overnight at 4° C. The wells were rinsed with PBS and blocked withPBS/1% (w/v) casein for 2 h at 37° C. Approximately 10¹² phage was addedto the blocked wells and incubated for 2 h at 37° C. After 10× washingwith PBS/0.1% (v/v) TWEEN 20, the bound phage was eluted with 0.1 Mtriethylamine, neutralized and mixed with exponentially growing TG1cells. Titration of eluted phage was performed and infected bacterialcells were superinfected with M13K07 and grown overnight at 37° C. Thepurified phage from the overnight culture was used as the input for thenext round of panning. The panning was continued for three more roundsfollowing the same procedure except that the amount of coatedRBD-fragments was reduced to 15 μg, 10 μg and 5 μg for the second, thirdand fourth rounds of panning, respectively.

Individual TG1 colonies obtained after round four of panning weresubjected to phage ELISA screening, essentially as described elsewhere(Doyle et al, 2008), with the exception that 5 μg/ml of toxin (TcdA andTcdB) and recombinant fragments (TcdA-RBD-f1 and TcdB-RBD-f1) werecoated onto microtiter plates. All positive clones were sent for DNAsequencing. Unique clones that gave high phage ELISA signals wereselected for large-scale expression and purification. Seven uniqueTcdA-specific and 7 unique TcdB-specific binders, all determined to beV_(H)Hs based on the presence of characteristic amino acids at positions42, 49, 50 and 52 (FIG. 2), were selected.

Example 4: Expression and Purification of Selected V_(H)Hs

The unique TcdA-specific and TcdB-specific binders of Example 3 weresub-cloned into expression plasmids for protein expression andpurification.

Phagemid vectors containing the DNA of selected V_(H)H clones werepurified using the QIAPREP MiniPrep Kit. Of the 14 V_(H)Hs, 11 cloneswere PCR amplified from the pMED1 phagemid vector with eitherBbsI1-V_(H)H (5′-TATGAAGACACCAGGCCCAGGTAAAGCTGGAGGAGTCT-3′ SEQ ID NO:63)or BbsI2-V_(H)H (5′-TATGA AGACACCAGGCCCAGGTGCAGCTGGTGGAGTCT-3′ SEQ IDNO:64) sense primers and BamHI-V_(H)H(5′-TTGTTCGGATCCTGAGGAGACGGTGACCTG-3′ SEQ ID NO:65) antisense primer.These PCR fragments were digested with BbsI and BamHI restrictionenzymes and ligated into the similarly digested pSJF2H expression vector(Arbabi-Ghahroudi et al, 2009b). Three of the 14 clones containedinternal BbsI or BamHI sites and were cloned into the pMED2 expressionvector via digestion with SfiI. The vector pMED2 is a modified versionof pSJF2H which contains SfiI restriction enzyme sites in its multiplecloning site. Since V_(H)H sequences in pMED1 are flanked with SfiIrestriction sites, no PCR amplification was required for sub-cloning.Upon ligation, all plasmids were transformed into electro-competent TG1E. coli and selected on LB agar plates+100 μg/ml ampicillin. Colonieswere screened by colony PCR for inserts and the DNA sequenced.

V_(H)Hs were expressed using the 5-day M9 minimal media method(Arbabi-Ghahroudi et al, 2009c). After induction of protein expression,cell cultures were harvested at 6,000 rpm×30 min (4° C.), thesupernatant decanted, and the periplasmic contents extracted from thecell pellet. Briefly, each pellet was resuspended in 30 ml of ice-coldTES buffer (0.2 M Tris-HCl buffer pH 8.0, 20% (w/v) sucrose, 0.5 mMEDTA) and incubated on ice for 30 min. Next, 40 ml of ice-cold ⅛ TES wasadded, incubated an additional 30 min on ice and the slurry centrifugedat 12,000 rpm for 30 min (4° C.). The resulting supernatant was dialysedovernight into IMAC buffer A (10 mM HEPES buffer pH 7.0, 500 mM NaCl)and purified as previously described (Hussack et al, 2009). Elutedfractions were analyzed by SDS-PAGE and Western blotting before beingdialysed into PBS. V_(H)H concentrations were determined by absorbancemeasurements at 280 nm using theoretical MW and extinction coefficientscalculated with the EXPASY ProtParam Tool(http://expasy.org/tools/protparam.html) (Pace et al, 1995).

The expression of the anti-TcdA and anti-TcdB V_(H)H was targeted to theperiplasm of TG1 E. coli and purified (FIG. 1E) with yields ranging from1.2-72.3 mg/I bacterial culture (Table 1, Example 7).

Example 5: Pentabody Expression and Purification

Two TcdA-specific pentameric V_(H)Hs (pentabodies) were constructed aspreviously described (Zhang et al, 2004b), using the vector pVT2 whichcontains the verotoxin B (VTB) subunit pentamerization domain.Pentabodies were expressed in TG1 E. coli, as described in Example 4,the cells were lysed and processed as previously described (Hussack etal, 2009), and proteins purified with HITRAP IMAC columns using animidazole gradient (0-500 mM) for elution. The pentabodies wereconstructed base on the highest affinity anti-TcdA V_(H)Hs, A5.1 andA20.1 (FIG. 3). The resulting pentabodies are referred to hereafter asA5.1p and A20.1p.

Example 6: Enzyme-Linked Immunosorbant Assay (ELISA)

ELISA experiments were used to characterize the binding of V_(H)Hmonomers and pentamers of Examples 4 and 5, as well as their ability torecognize natural and recombinant antigen.

ELISA was used to determine if the purified anti-toxin V_(H)H monomersrecognized native TcdA or TcdB and recombinant TcdA-RBD-f1 orTcdB-RBD-f1 fragments. Equivalent molar concentrations of proteins (BSA,TcdA, TcdB, TcdA-RBD-f1, and TcdB-RBD-f1) were coated overnight in 96well microtiter plates at 4° C. The next day, wells were blocked with 3%(w/v) skim milk diluted in PBS-T. After washing with PBS-T, purifiedV_(H)Hs at concentrations as high as 10 μg/ml were added to wells withthe various coated antigens for 1 h at 37° C. Wells were washed withPBS-T, rabbit anti-His₆ IgG conjugated with HRP (1:2,500 in PBS) addedfor 1 h at room temperature and the wells were washed an additional 5×with PBS-T. Rabbit anti-His₆ IgG-HRP did not recognize the N-terminalHis₆ epitope tags on recombinant RBD fragments (data not shown). Bindingwas detected with TMB substrate (KPL), the reactions were stopped with 1M H₃PO₄ and absorbance read at 450 nm. All conditions were performed intriplicate and the reported values are representative of two independentexperiments.

ELISA demonstrated that 6 of 7 anti-TcdA V_(H)Hs recognized native TcdAand TcdA-RBD-f1 and that none of the V_(H)Hs cross-reacted with TcdB orTcdB-RBD-f1 (FIG. 4A). Of the 7 anti-TcdB V_(H)Hs tested, 4 recognizedTcdB and TcdB-RBD-f1 and one clone (B5.2) also recognized TcdA (FIG.4B). ELISA experiments were also performed by coating 5 μg/ml of TcdA inmicrotiter wells and serially diluting V_(H)Hs from 10 μg/ml to 1 ng/ml(FIGS. 4C-D). The remaining detection steps were performed as describedabove. With this ELISA, which was performed at a higher V_(H)Hconcentration, a fifth V_(H)H, B15.3, was shown to bind to TcdB.

A second ELISA was used to compare the activities of monomeric andpentameric V_(H)Hs for TcdA. TcdA (5 μg/ml) was coated overnight in 96well microtiter plates and blocked with 3% milk, before addition ofequivalent molar concentrations of V_(H)H monomers and pentamers. V_(H)Hbinding to TcdA was detected with HRP-labelled rabbit-anti-His₆ IgG andTMB substrate. Binding of the pentamers to immobilized TcdA by ELISA wassimilar to monomeric versions of the same V_(H)Hs (FIG. 3).

Example 7: Size Exclusion Chromatography and Surface Plasmon ResonanceAnalysis

The affinity of the TcdA- and TcdB-specific V_(H)Hs of Example 4 totheir respective antigens was determined by surface plasmon resonance(SPR).

Size exclusion chromatography was performed on all purified V_(H)Hsusing a SUPERDEX 75 size exclusion column (GE Healthcare) as previouslydescribed (To et al, 2005) under the control of an AKTA FPLC. Briefly,V_(H)Hs were applied at concentrations ranging from 0.75-1 mg/ml (≅45-60μM) with a flow rate of 0.5 ml/min in a mobile phase that consisted ofHBS-EP running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and0.005% (v/v) P20 surfactant). As expected, all were non-aggregatingmonomers (FIG. 5).

Fractions from the size exclusion column were then used for affinitymeasurements. The binding kinetics for the interaction of anti-toxinV_(H)Hs and TcdA or TcdB were determined by surface plasmon resonanceusing a BIACORE 3000 biosensor system (GE Healthcare). A total of 10,377resonance units (RUs) of TcdA and 5,980 RUs of mouse IgG 13D9 control(Liu et al, 2000) were immobilized on a CM5 sensor chip (GE Healthcare).Anti-TcdA V_(H)H affinity measurements were carried out in HBS-EPrunning buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005%(v/v) P20 surfactant) at a flow rate of 40 μl/min. Surfaces wereregenerated by washing with either running buffer or 10 mM glycine pH2.0. Initial attempts to immobilize TcdB directly onto a CM5 sensor chipwere unsuccessful due to the toxin's low pl (theoretical pl=4.42). TcdBwas therefore biotinylated with the EZ-LINK Sulfo-NHS-LC-LC-Biotin kitfrom Pierce (Rockford, Ill.) and 825 RUs were immobilized onto astreptavidin-coated CM5 sensor chip. However, due to the size differenceof TcdB-biotin (269 kDa) compared to streptavidin (53 kDa), not allstreptavidin sites were occupied and roughly 1 TcdB-biotin wasimmobilized for every 7 streptavidin molecules. Furthermore, no bindingwas observed between the anti-TcdB V_(H)Hs and immobilized TcdB-biotin.Data on the V_(H)H-TcdB-RBD-f1 interaction was collected by immobilizingTcdB-specific V_(H)Hs onto the CM5 sensor chip (RUs ranging from 215 to1209) and injecting TcdB-RBD-f1 at 20 μl/min. The IgG 13D9 or humansingle-domain antibody HVHP420 (To et al, 2005) served as controls. Inall cases, data were analyzed with BIAevaluation 4.1 software (GEHealthcare).

Surface plasmon resonance (SPR) analysis revealed 6 of 7 anti-TcdAV_(H)Hs specifically bound TcdA with equilibrium dissociation constantsranging from 290 nM for A19.2 to 2 nM for A20.1 (FIG. 6). ObservedV_(H)H affinities for TcdA were strong, with four of the clones havingK_(D) values ranging from 2 to 24 nM (Table 1). The anti-TcdA V_(H)Hsisolated here are among the highest affinity proteinaceous toxin-bindingsingle-domain antibodies characterized to date (Stewart et al, 2007;Goldman et al, 2006; Liu et al, 2007a; Hmila et al, 2008; Goldman et al,2008; Anderson et al, 2008).

Analyzing the TcdB-binding V_(H)Hs by SPR was more challenging. Initialattempts to immobilize TcdB onto the CM5 dextran biosensor chip may havebeen hindered by the low theoretical pl of TcdB. An attempt tobiotinylate TcdB for immobilization on a streptavidin-coated biosensorchip was equally as unsuccessful. To circumvent this problem, anti-TcdBV_(H)Hs were immobilized directly onto the CM5 dextran chips and datacollected using various concentrations of TcdB-RBD-f1. Analyzable datacould only be collected for 3 of 7 anti-TcdB V_(H)Hs, with affinityconstants ranging from 100 nM to 400 nM (FIG. 6; Table 1). Specificbinding was detected for the other 4 anti-TcdB V_(H)Hs, however, thedata was non-analyzable (FIG. 7).

TABLE 1 Properties of anti-C. difficile toxin A and B V_(H)Hsingle-domain antibodies. Ex- pression MW Yield k_(on) K_(D) Neutral-V_(H)H (kDa) pl^(a) (mg/l) (M⁻¹s⁻¹) _(off)(s⁻¹) (nM) izing? A1.3 16.756.71 1.4 NB NB NB No^(b) A4.2 15.73 8.59 31.3 6.7 × 10⁵ 1.6 × 10⁻² 24Yes A5.1 15.80 6.71 55.5 1.6 × 10⁶ 5.0 × 10⁻³ 3 Yes A19.2 16.01 8.61 3.81.4 × 10⁴ 3.9 × 10⁻³ 290 Yes A20.1 16.61 6.64 72.3 8.2 × 10⁵ 1.6 × 10⁻³2 Yes A24.1 16.71 6.71 8.5 6.0 × 10⁴ 1.6 × 10⁻² 260 Yes A26.8 16.02 6.6564.9 1.4 × 10⁶ 1.6 × 10⁻² 12 Yes B5.2 15.11 6.04 6.7 2.0 × 10³ 2.0 ×10⁻⁴ 100 No^(b) B7.3 16.13 8.93 1.5 NB NB NB NA B13.2 15.79 7.98 4.0 NBNB NB NA B13.3 15.62 8.00 1.6 NB NB NB NA B13.6 15.01 8.00 3.6 2.5 × 10³1.0 × 10⁻³ 400 No B15.3 15.58 8.59 1.2 NB NB NB NA B15.5 15.23 7.18 4.62.8 × 10³ 1.0 × 10⁻³ 357 No^(b) ^(a)Theoretical pl calculated using theEXPASY ProtParam tool (expasy.ch/tools/protparam.html). ^(b)Notneutralizing at concentration as high as 1 μM. NB: no binding detectedby BIACORE. NA: not attempted.

Example 8: In Vitro Toxin Neutralization Assay

The human lung fibroblast (HLF) cell cytotoxicity assay is routinelyused for analyzing the presence of C. difficile toxins in biologicalsamples (Babcock et al, 2006). The assay was used here to determine ifanti-toxin V_(H)Hs of Example 4 or 5 could neutralize the cytopathiceffects of TcdA and TcdB.

HLF cells (ATCC#CCL-186) were purchased from ATCC (Manassas, Va.) andmaintained in Eagle's minimal medium (Invitrogen, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (Invitrogen) at 37° C. with 5%CO₂. Cells were seeded in sterile 96 well microtiter plates (2×10⁴ cells200 μl⁻¹ well⁻¹) for 20 h, allowing for the formation of confluentmonolayers.

Initially, a dose-response experiment was conducted to find the minimumconcentration of TcdA and TcdB which induced 100% HLF cell roundingafter 24 h post toxin addition. To do so, 10 μl of sterile filtered TcdAor TcdB were added to wells containing confluent monolayers, givingfinal toxin concentrations ranging from 500 ng/ml to 0.5 ng/ml. Eachconcentration was performed in triplicate and the assay performed twice.HLF cells were scored visually for rounding at various time points over24 h. For all subsequent assays, 100 ng/ml of TcdA and 20 ng/ml of TcdBwere used.

For experiments involving V_(H)Hs, 20 μl of purified and sterilefiltered monomeric or pentameric V_(H)Hs were added to HLF cells with 10μl TcdA/B or 10 μl PBS. For experiments involving combinations of 2 or 3V_(H)Hs, the concentration of each V_(H)H was reduced by ½ and ⅓,respectively, giving the same final concentrations as experimentsinvolving a single V_(H)H. Importantly, V_(H)Hs and toxin were notpre-incubated; rather, each was added directly to HLF monolayers attime=0 h; this was more representative of in vivo scenarios and did notbias the in vitro results by pre-incubating. HLF cells containing PBS,V_(H)H, toxin, or V_(H)H+toxin were scored visually for cell roundingusing a confocal microscope at 8 h and 24 h post antibody/toxinaddition. Assays were performed in triplicate and repeated twice. Eachassay was performed on fresh preparations of HLF cells (passage 3-5) andV_(H)Hs were from separate purifications. The purified TcdA and TcdBstock remained the same for all assays.

Human lung fibroblast (HLF) cytotoxicity assays were used to determinewhether V_(H)Hs could neutralize TcdA- or TcdB-induced HLF cellrounding. Dose-response experiments with TcdA (FIG. 8A) and TcdB (FIG.8B) determined the minimum toxin concentrations capable of 100% cellrounding after 24 h to be 50 ng/ml and 5 ng/ml, respectively. For allsubsequent experiments, 2× this minimum concentration was used (i.e.,100 ng/ml of TcdA and 10 ng/ml of TcdB). V_(H)Hs had no effect on HLFcells when incubated in the absence of toxin A (FIG. 8F). When V_(H)Hsand TcdA were added simultaneously to HLF cells, 6 of 7 anti-TcdAV_(H)Hs inhibited TcdA-induced cell rounding in a dose-dependent mannerat 8 h (data not shown) and 24 h post TcdA addition (FIG. 8C-F). Theneutralizing capacity of the 4 strongest monomeric V_(H)Hs (A4.2, A5.1,A20.1 and A26.8) was similar at all concentrations tested, reflective oftheir close range of K_(D)'s (2-24 nM). The weakest neutralizers, A19.2and A24.1, also possessed the weakest affinity constants of 290 and 260nM, respectively. The non-binding A1.3 V_(H)H did not inhibit cellrounding.

The toxin neutralizing efficacy of various combinations of V_(H)Hs wastested. All possible pair-wise and triplet combinations were tested.When various combinations of A4.2, A5.1, A20.1 and A26.8 were tested,their TcdA neutralizing efficacy was greater than any of the V_(H)Hsalone. These observations suggested the V_(H)Hs recognized distinctepitopes on TcdA, which was subsequently confirmed for A20.1 byco-injection BIACORE surface plasmon resonance (SPR) epitope mappingexperiments (FIG. 10). In contrast, the other potent neutralizersappeared to bind to overlapping epitopes on TcdA. These data explain theincreased neutralizing capacity seen for pairs and triplet combinationscontaining A20.1, but do not explain why some pairs (i.e., A5.1/A26.8)or triplet combinations (i.e., A4.2/A5.1/A26.8) show greater efficacythan the individual V_(H)Hs. The BIACORE SPR data indicated a 1:1binding stoichiometry, which is difficult to reconcile with theobservation of enhanced neutralizing efficacy with mixed V_(H)Hs bindingto overlapping epitopes. The binding stoichiometry determination assumesa mainly active toxin surface, which may not be the case since the toxinpreparations showed breakdown products (FIG. 1B and FIG. 9B/C).

Pentabodies A5.1p and A20.1p, were also tested for toxin neutralizingefficacy. Surprisingly, the pentabodies showed similar neutralizingefficacy to their monomeric counterparts at the same concentration (datanot shown). While it is possible that size effects increased thehindrance of TcdA binding to cell receptors, this effect on neutralizingpotency may have been offset with a reduction in effective V_(H)Hconcentration. For example, if only 1 of 5 V_(H)H molecules could bindTcdA, the number of total V_(H)Hs available for TcdA binding would bereduced by 5-fold.

The neutralizing capacity of the anti-TcdB V_(H)Hs was also tested. Noneof the 3 TcdB-specific V_(H)Hs were capable of TcdB neutralization, evenat a concentration of 1 μM (data not shown). It is not clear whether theV_(H)H affinity was insufficient for neutralization, or if the epitopesthese V_(H)Hs were raised against was not capable of preventing TcdBbinding to fibroblast cell receptors. SPR Data was only collected forthe TcdB-RBD-f1-V_(H)H interaction; it is possible that the V_(H)Haffinities for TcdB may be considerably lower and this could account forpoor neutralization.

The structure of TcdA-RBD contains 7 putative carbohydrate binding sites(Greco et al, 2006; Ho et al, 2005), which are thought to interact withepithelial cell surface receptors to mediated endocytosis (Florin andThelestam, 1983). Due to geometric constraints, all 7 sites cannotaccess the cell-surface receptors simultaneously, although multiple (<7)low affinity interactions are predicted (Greco et al, 2006). As such,avidity appears to be crucial to the strength of toxin binding to itscellular receptors. It was hypothesized that pooling of neutralizingV_(H)Hs which recognized distinct epitopes on TcdA-RBD may enhanceneutralizing potency through greater hindrance of toxin-cell receptorcontacts. When various combinations of A4.2, A5.1, A20.1 and A26.8 weretested, their TcdA neutralizing efficacy was greater than any of theV_(H)Hs alone. These observations suggested the V_(H)Hs recognizeddistinct epitopes on TcdA, which was subsequently confirmed for oneV_(H)H (A20.1) by co-injection BIACORE epitope mapping experiments(Example 9).

Example 9: Epitope Mapping

To gain insight into whether the TcdA-specific V_(H)Hs recognized alinear or conformational epitope on TcdA, and whether the V_(H)Hs couldbind unique, non-overlapping epitopes, a combination of Westernblotting, ELISA, and SPR was used.

Western blots using both denaturing SDS-PAGE and native PAGE, andcontaining TcdA were probed with anti-TcdA V_(H)Hs or control anti-TcdAIgG (PCG4; Novus Biologicals, Littleton, Colo.) to determine if theV_(H)Hs recognized linear or conformational epitopes. For denaturingSDS-PAGE Western blots, TcdA (0.75 μg/lane), A4.2 V_(H)H (1 μg/lane) andPCG4 IgG (1 μg/lane) were separated on 12.5% SDS-PAGE gels underreducing conditions and transferred to PVDF membranes at 100 V for 1 h.Membranes were blocked for 1 h with 5% (w/v) BSA diluted in PBS-Tfollowed by probing with various V_(H)Hs (25 μg/10 ml blocking buffer)or PCG4 (10 μg/10 ml blocking buffer) for 1 h. Membranes were washed 4×in PBS-T followed by addition of either: (i) mouse anti-His₆IgG-alkaline phosphatase (AP) conjugate (Abcam, Cambridge, Mass.)diluted 1:5,000 in blocking buffer, (ii) HisDetector Nickel-AP conjugate(Mandel Scientific, Guelph, ON, Canada) diluted 1:5,000 in blockingbuffer, or (iii) goat anti-mouse IgG-AP conjugate (Cederlane,Burlington, ON, Canada) diluted 1:10,000 in blocking buffer for 1 h.After a final set of 4 washes, membranes were subjected to AP substrate(BioRad, Hercules, Calif.) for 7 min, washing in dH₂O and air drying.For native PAGE Western blots, TcdA, V_(H)H and PCG4 (concentrations asabove) were separated on 8% PAGE gels (without SDS) at 100 V for 2 h onice. Next, gels were transferred to PVDF membranes at 20 V for 14 h at4° C. Membranes were blocked, probed, washed and detected using the sameprotocol as for SDS-PAGE Western blots.

Only A19.2 V_(H)H recognized TcdA run under denaturing/reducingconditions (FIG. 9B). The anti-TcdA mAb PCG4 (Lyerly et al, 1986), whichwas previously shown to recognize TcdA in Western blots (Ochsner et al,2009), confirmed that TcdA was transferred to the blot. The weak signalobtained from A19.2 relative to PCG4 was likely due to the low affinityand/or lack of avidity of A19.2 for TcdA. In the absence of primaryantibody, the secondary conjugates Nickel-AP and goat anti-mouse IgG-APdid not bind TcdA as expected. The V_(H)H A4.2 and PCG4 were included toconfirm the functionality of the secondary conjugates. Undernon-denaturing conditions (native PAGE), V_(H)H binding to TcdA wasoriginally probed with anti-His₆ IgG-AP and this secondary antibody wasfound to cross-react with TcdA in the absence of V_(H)H (data notshown). To overcome this, the secondary antibody was replaced withNickel-AP. Using this secondary conjugate, the V_(H)Hs A4.2, A5.1, A20.1and A26.8 recognized native TcdA while the non-binding A1.3 essentiallydid not react with TcdA (FIG. 9C). The Nickel-AP secondary conjugate didnot bind native TcdA in control blots. The diffuse signal and poormigration pattern of A4.2 V_(H)H control is likely due to its high pl(theoretical pl: 8.59). To confirm the presence of TcdA on native PAGEblots, PCG4 was used as a control. Just like anti-His₆ IgG-AP, thesecondary antibody goat anti-mouse IgG-AP also bound TcdA in the absenceof the primary probe, PCG4 in this case (FIG. 9C).

To further investigate whether the V_(H)Hs recognized linear orconformational epitopes, ELISA was performed with TcdA exposed tovarious temperature above and below its thermal unfolding midpointtemperature (T_(m)˜55° C.; Salnikova et al, 2008). Briefly, TcdA (5μg/ml) was exposed to the following condition for 30 min: 4° C., 20° C.,37° C., 50° C., 60° C. or 70° C. After temperature treatment, 100 μl ofTcdA was coated in 96 well microtiter plates overnight at 4° C. and theassay performed essentially as described in Example 6, except that0.05-1 μg/ml of V_(H)H was used. All conditions were performed induplicate and the reported values are representative of two independentexperiments.

For 4 of the 5 V_(H)Hs, A4.2, A5.1, A20.1 and A26.8, binding to TcdA wascompletely abolished when TcdA was heated above its T_(m) (FIG. 9A),confirming the above results that the V_(H)Hs recognize a conformationalepitope on TcdA. A19.2 was found to bind TcdA with the same strength atall temperature, indicating that this V_(H)H recognizes a linearepitope. The epitopes must be located in the region of TcdA responsiblefor cell-receptor binding, since llama immunization and library panningwere both performed with TcdA-RBD-f1 and because the V_(H)Hs were shownto recognize this recombinant fragment by ELISA (FIG. 4A).

BIACORE surface plasmon resonance (SPR) co-injection experiments werealso used to determine if the V_(H)Hs could bind unique, non-overlappingepitopes on TcdA. Briefly, 80 μl of the first V_(H)H diluted in HBS-EPbuffer to a concentration of 20× its K_(D) was injected over 10,287 RUsof immobilized TcdA at 40 μl/min. Following injection of the firstV_(H)H, buffer or a second V_(H)H (80 μl total volume, at 20× K_(D)) wasinjected at 40 μl/min over the TcdA surface already saturated with thefirst V_(H)H. Data were collected on all possible paired combinations ofA4.2, A5.1, A20.1 and A26.8, in both orientations (i.e., each V_(H)Hacted as the first and second V_(H)H). Data were collected and evaluatedas described in Example 7.

The observation that combining anti-TcdA V_(H)Hs increased TcdAneutralizing efficacy relative to single V_(H)Hs at the sameconcentration (FIG. 8C-E; Example 8) suggested the antibodies recognizeddistinct, non-overlapping epitopes. Co-injection BIACORE SPR experimentswere performed with pairs of V_(H)Hs, in both orientations, to determineif antibodies could bind TcdA simultaneously (FIG. 10). Of the pairedcombinations, only those involving A20.1 V_(H)H showed a significantincrease in response consistent with theoretical R_(max) values(˜160-180 RUs) upon co-injection. This suggests that A20.1 is free tobind TcdA when A4.2, A5.1 or A26.8 are bound at saturatingconcentrations and also indicates the A20.1 epitope is distinct and doesnot hinder binding of the other three V_(H)Hs. For A4.2, A5.1 and A26.8,however, only minor changes in response were seen upon co-injection withtheoretical R_(max) values not reached, an indication that the V_(H)Hswere binding overlapping epitopes and hindering their binding to TcdA(FIG. 10). This was confirmed by co-injection of all three of theseV_(H)Hs simultaneously (FIG. 11).

Taken together, the BIACORE SPR-based epitope mapping studies suggestA20.1 freely binds TcdA at a site that does not overlap with, or is notsterically hindered by, A4.2, A5.1 or A26.8 binding. These latter threeV_(H)Hs bind at sites on TcdA that hinder freely accessible binding ofthe others, suggesting these antibodies share a single epitope, or bindepitopes in such close proximity to one another that it preventsunhindered interaction with TcdA. It is likely that “overlapping”V_(H)Hs recognize repeating epitopes. With mixed V_(H)H, the spatialgeometry of binding along the toxin RBD length at multiple epitope sitesis such that it makes them more effective toxin neutralizers than when asingle V_(H)H is bound. Thus, despite binding an overlapping epitope,the mixture of V_(H)H geometries would enhance steric effects (andneutralizing capacity) compared to single V_(H)H species which presentsa single geometry.

Using BIACORE SPR, inhibition of the V_(H)H-TcdA interaction with twotrisaccharides known to bind TcdA-RBD was also attempted, in order togain insight into the TcdA epitope recognized by the V_(H)Hs. CD-grease(α-Gal-(1,3)-β-Gal-(1,4)-β-GlcNAcO(CH₂)₈CO₂CH₃; Greco et al, 2006) andLe^(X)-AmHex (Gal-β1,4-(Fuc-α1,3)-GlcNAc-(CH₂)₆—NH₂—HOAc; P. Zhang andC. C. Ling, unpublished) are known to interact with the carbohydratebinding pockets of TcdA-RBD (Greco et al, 2006). Briefly, 4 V_(H)Hs(i.e., A4.2, A5.1, A20.1 and A26.8) at K_(D) concentrations wereinjected alone or in the presence of trisaccharide (2 mM) overimmobilized TcdA (8000 RUs). The response obtained from the interactionof trisaccharide with TcdA was subtracted from response generated byV_(H)H+trisaccharide co-injection experiments. Then, the response ofeach V_(H)H to TcdA in the presence of trisaccharide was compared to theresponse generated by the V_(H)H-TcdA interaction. FIG. 12 shows arepresentative example in which CD-grease and Le^(X)-AmHextrisaccharides are free to bind TcdA in the presence of A26.8 V_(H)H. Wealso show in FIG. 13 two examples of CD-grease binding freely to TcdA inthe presence of saturating amounts of bound V_(H)Hs A20.1 (FIG. 13A) andA26.8 (FIG. 13B). Collectively, these studies indicate the V_(H)Hs arenot binding to TcdA in such a way as to prevent trisaccharide binding.Therefore, the anti-TcdA V_(H)H tested do not bind in the carbohydratebinding pocket, or sufficiently close to it, as they do not inhibit thefree trisaccharide from accessing the toxin.

V_(H)Hs have long, flexible CDR3 loop regions that have been shown toform a convex paratope that can extend into clefts or active sites ofprotein antigens (De Genst et al, 2006). The recently solved crystalstructure of TcdA-RBD was shown to contain seven carbohydrate bindingpockets thought to be involved in cell receptor binding (Greco et al,2006). The methods just described were to investigate whether TcdAneutralization was due to V_(H)H binding in the TcdA-RBDcarbohydrate-binding pocket. The binding of the neutralizing V_(H)Hs toTcdA was not inhibited in the in co-injection experiments (FIG. 12).Both CD-grease and Le^(X)-AmHex trisaccharides were used at theirpredicted K_(D) concentrations and were shown to bind immobilized TcdAby BIACORE SPR, but did not prevent V_(H)H binding to TcdA when thetrisaccharide response for TcdA was subtracted from response generatedby co-injection experiments (FIG. 12). In additional BIACORE SPRexperiments, both of the trisaccharides were found to be free to bindV_(H)H-saturated TcdA (data not shown). Furthermore, V_(H)H binding toTcdA was not inhibited in ELISA with trisaccharide concentrations ashigh as 10 mM (data not shown). Taken together, these data suggest thepresent V_(H)Hs do not inhibit free trisaccharides from accessing theirbinding sites on TcdA-RBD and that the V_(H)Hs are not binding at sitesoccupied by the trisaccharides.

Example 10: Engineering, Expression, and Purification of Mutant V_(H)Hs

The extreme pH and protease-rich environment of the uppergastrointestinal tract is a major obstacle facing orally-administeredprotein therapeutics, including antibodies. For these reasons, theV_(H)Hs of Example 4 were engineered to improve biophysical properties.The methods used herein are also summarized in Hussack et al (2011).

A panel of C. difficile toxin A (TcdA)-specific V_(H)Hs were expressedwith an additional disulfide bond by introducing Ala/Gly⁵⁴→Cys⁵⁴ andIle⁷⁸→Cys⁷⁸ mutations. It was hypothesized that the addition of adisulfide bond in the hydrophobic core of a V_(H)H antibody fragmentsbetween framework 2 (FR2) and FR3 would not only increase thermalstability at neutral pH, as previously reported (Hagihara et al, 2007;Saerens et al, 2008), but would also impart resistance to proteolyticdegradation and increase antibody stability at low pH. The sequences ofthe mutant V_(H)Hs are shown in FIG. 14.

To construct mutant V_(H)Hs with a second internal disulfide bond,splice-overlap extension (SOE) PCR was performed essentially asdescribed (Ho et al, 1989; Arbabi et al, 2010) using 4 primers for eachV_(H)H and two rounds of PCR. Nucleotides encoding amino acid residuesat positions 54 and 78 (IMGT numbering system) were changed toCys-coding nucleotides through primer-forced mutation. In the first PCR,two mutagenized overlapping sub-fragments were generated for eachV_(H)H. The primer pairs used for each V_(H)H were as follows: A4.2m(BbsI-VHH and A4.2mR-Cys, A4.2mF-Cys and BamHI-VHH); A5.1m (BbsI-VHH andA5.1mRCys, A4.2mFCys and BamHI-VHH); A19.2m (BbsI-VHH and A19.2mR-Cys,A19.2mF-Cys and BamHI-VHH); A20.1m (A20.1mSfiI-F and A20.1mR-Cys,A20.1mF-Cys and A20.1mSfiI-R); A24.1m (A20.1mSfiI-F and A24.1mR-Cys,A24.1mF-Cys and A20.1mSfiI-R); A26.8m (BbsI-VHH and A26.8mR-Cys,A26.8mF-Cys and BamHI-VHH). Each sub-fragment was gel purified andspliced with its partner fragment in a second PCR. Briefly, 160 ng ofeach sub-fragment were added to a 50 μl PCR mixture containing Pfu DNApolymerase, dNTPs and reaction buffer. The reaction was placed in athermal cycler and the two fragments were spliced together using aprogram consisting of a preheating step at 94° C. for 5 min and 10cycles of 94° C. for 30 s, 55° C. for 30 s, and 72° C. for 1 min. Toamplify the spliced products, the reaction was heated to 94° C. for 3min, 5 pmol (0.5 μl) of each primer pair was added (BbsI-VHH andBamHI-VHH for A4.2m, A5.1m, A19.2m, and A26.8m; A20.1mSfiI-F andA20.1mSfiI-R for A20.1m and A24.1m), and 35 PCR cycles were performedexactly as described above. The resulting fragments were gel purified,digested with BbsI and BamHI (A4.2m, A5.1m, A19.2m, and A26.8) or SfiI(A20.1m and A24.1m) restriction enzymes and ligated into similarlydigested expression vectors (pSJF2H or pMED2). All mutant V_(H)Hs wereexpressed in the same vectors as their counterpart wild-type V_(H)Hs(see Example 4). For cloning into pSJF2H, mutant V_(H)H DNA wereamplified with primers containing 5′ BbsI and 3′ BamHI restriction siteswhile cloning into pMED2 required amplification with primers containingboth 5′ and 3′ SfiI restriction sites. The vectors were transformed intoTG1 E. coli for V_(H)H expression. Positive colonies were identified bycolony-PCR and DNA sequencing, using the M13RP and M13FP primers.

TABLE 2 Primers used for construction of disulfide mutant V_(H)Hs.Primers Sequence (5′ → 3′) Purpose M13FP GTA AAA CGA CGG CCA  ScreeningGT (SEQ ID NO: 66) M13RP CAG GAA ACA GCT ATG ScreeningAC (SEQ ID NO: 67) BbsI-VHH^(a) TAT GAA GAC ACC AGG  ConstructingCCC AGG TAA AGC TGG mutants AGG AGT CT (SEQ ID NO: 63) BamHI-VHH^(a)TTG TTC GGA TCC TGA Constructing GGA GAC GGT GAC CTG mutants(SEQ ID NO: 65) A4.2mR-Cys AGT CTG CAT AGT ATG ConstructingTGC TAC CAC CAC TCC A4.2m GGC TAA CAG CGC AAA CAA ACT C (SEQ ID NO: 68)A4.2mF-Cys TAG CAC ATA CTA TGC Constructing AGA CTC CGT GAA GGG4.2m/A5.1m CCG ATT CAC CTG CTC CAG AGA C (SEQ ID NO: 69) A5.1mR-CysAGT CTG CAT AGT ATG Constructing TGC TAC TAC CAT TCC A5.1mGGG TAA TAA CGC ATA CAA ACT C (SEQ ID NO: 70) A19.2mR-CysACT CTA CAT AGG CAC Constructing TAT TAC CAC CAC GCC A19.2mGGC TAA TAC CGC ATA CAA ACT C (SEQ ID NO: 71) A19.2mF-CysTAA TAG TGC CTA TGT Constructing AGA GTC CGT GAA GGG A19.2mCCG ATT CAC CTG CTC CAG AGA C (SEQ ID NO: 72) A20.1mSfil-FACC GTT GCG CAG GCC Constructing CAG CCG GCC ATG GCC A20.1m/A24.1mCAG GTA CAG C (SEQ ID NO: 73) A20.1mR-Cys TGT CTG CAT AGT ATGConstructing TGG TCC GCC CCG TAG A20.1m AAC TCC CCG CGC ATA CAA ACT C(SEQ ID NO: 74) A20.1mF-Cys GAC CAC ATA CTA TGC ConstructingAGA CAG CGT GAA GGG A20.1m CCG ATT CAC CTG CTC CAG AGA C (SEQ ID NO: 75)A20.1mSfil-R GTT CGG ATC CCT GGC Constructing CGG CCT GGC CTG AGGA20.1m/A24.1m AGA CGG TGA CC (SEQ ID NO: 76) A24.1mR-CysAGT CTG CAT AGC GTG Constructing TGC TAC CTC CAC CCC A24.1mAGC TAA TAC CGC ATA CAA ACT C (SEQ ID NO: 77) A24.1mF-CysTAG CAC ACG CTA TGC Constructing AGA CTC CGT GAA GGG A24.1mCCG ATT CAC CTG CTC CAG AGA C (SEQ ID NO: 78) A26.8mR-CysAGT CTG CAT AGT ATG Constructing TGC TCG TAC CAG TCG A26.8mAGC TAA TAA CGC ATA CAA ACT C (SEQ ID NO: 79) A26.8mF-CysGAG CAC ATA CTA TGC Constructing AGA CTC GGT GAA GGG A26.8mCCG GTT CAC CTG CTC CAG AGA C (SEQ ID NO: 80) ^(a)Reverse and forwardprimers for construction of A4.2m, A5.1m, A19.2m, and A26.8m.

Expression and purification of mutant V_(H)Hs was performed as describedin Example 4, followed by dialysis into phosphate-buffered saline pH 7.3(PBS), into distilled, deionized water (ddH₂O) for mass spectrometry(MS) analysis, or into 10 mM phosphate buffer pH 7.3 for CD experiments.Soluble mutant V_(H)Hs were extracted from the periplasm of TG1 E. coliand purified by immobilized-metal affinity chromatography (IMAC) withpurified yields ranging from 3-12 mg/I of bacterial culture.Non-reducing SDS-PAGE and Western blot analysis of the purified productsrevealed the mutant V_(H)Hs were of high purity and did not forminterdomain disulfide bonds (FIG. 15A). On non-reducing SDS-PAGE gels,mutant V_(H)Hs consistently ran slower than their correspondingwild-type V_(H)Hs (FIG. 15B).

Formation of the non-canonical disulfide linkages was confirmed by massspectrometry analysis on cyanogen bromide+trypsin digested mutantV_(H)Hs by identifying peptides containing the introduced disulfidebond. Briefly, 100 μl reactions containing 50 μg of mutant V_(H)H(diluted in PBS), 10 μl of 1 M HCl and 40 μL of CNBr (10 mg/ml stockprepared in 1 M HCl) were digested for 14 h at ambient temperature inthe dark. The next day, 100 μl of 1 M Tris-HCl, pH 8.6, and 60 μl oftrypsin (100 μg/ml stock; sequencing grade, Roche, Mississauga, ON,Canada) were added directly to the CNBr reaction mixture and incubatedfor 2 h at 37° C. Samples were then analyzed by non-reducing SDS-PAGE toensure digestion prior to MS analysis. An aliquot of the proteolyticdigest of each V_(H)H was re-suspended in 0.1% formic acid (aq) andanalyzed by nano-flow reversed-phase HPLC mass spectrometry(nanoRPLC-ESI-MS) using a nanoAcquity UPLC system coupled to a Q-TOFULTIMA hybrid quadrupole/TOF mass spectrometer (Waters, Milford, Mass.)with data dependent analysis (DDA). The peptides were first loaded ontoa 180 μm I.D.×20 mm 5 μm SYMMETRY C18 trap (Waters), then eluted to a100 μm I.D.×10 cm 1.7 μm BEH130C18 column (Waters) using a lineargradient from 0% to 36% solvent B (ACN+0.1% formic acid) in 36 min,36%-90% solvent B for 2 min. Solvent A was 0.1% formic acid in water.The peptide MS² spectra were searched against the V_(H)H proteinsequences using the MASCOT database searching algorithm (Matrix Science,London, UK). The MS² spectra of the disulphide-linked peptides weredeconvoluted using the MaxEnt 3 program (Waters) for de-novo sequencing.

To precisely confirm the presence of the introduced disulfide bond,mutant V_(H)Hs were digested with CNBr and trypsin (FIG. 16A) and theirdigests subjected to MS² analysis. The identification coverage of themutant V_(H)Hs from the analysis of their CNBr/trypsin digests usingnanoRPLC-ESI-MS with DDA was more than 30%. The disulfide-linked peptideions appeared prominent in the survey scan of the DDA experiment whenthe proteins were digested with a combination of CNBr and trypsin.Peptide fragments linked by the engineered Cys⁵⁴-Cys⁷⁸ disulfide bond(shown in bold text in FIG. 14) were positively identified for allmutant V_(H)Hs by manual de-novo sequencing (Table 3). For example, theprotein sequence coverage of A5.1m was 43% and a prominent ion at m/z526.25 (3+) was sequenced as a disulfide-linked peptide EFVCVITR (P1)and FTCSR (P2) as shown (FIG. 16B, FIG. 14, Table 3). An almost completedisulfide-linked y fragment ion series was observed from one peptidewith the other peptide attached as a modification via a disulfide bond,which remains intact under collision induced dissociation (CID) [59].

TABLE 3 Disulfide linkage determination of mutant V_(H)Hsby MS² analysis. Mutant V_(H)Hs were digested withCNBr and trypsin and the peptides analyzed byMS². The peptides containing the Cys⁵⁴-Cys⁷⁸disulfide linkage are shown with connectingcysteines bolded. A nearly perfect match betweenMW_(for) and MW_(exp) equates to the presence of theCys⁵⁴-Cys⁷⁸ disulfide linkage. MW_(for): formula(expected) molecular weight (Da);MW_(exp): experimental molecular weight (Da); ΔMW = MW_(for) - MW_(exp).V_(H)H CNBr/tryptic peptides MW_(for) MW_(exp) ΔMW  A4.2m EFVCAVSR FTCSR1519.69 1519.70 -0.01 (SEQ ID NO: 81 and 87)  A5.1m EFVCVITR FTCSR1575.75 1575.76 -0.01 (SEQ ID NO: 82 and 87) A19.2m EFVCGISR FTCSR1519.69 1519.64  0.05 (SEQ ID NO: 83 and 87) A20.1m EFVCAGSSTGR FTCSR1722.74 1722.84 -0.10 (SEQ ID NO: 84 and 87) A24.1mEFVCGISTNGGGSTR FTCSR 2064.91 2064.98 -0.07 (SEQ ID NO: 85 and 87)A26.8m EFVCVISSIGTSTYYADSVK 2766.25 2766.33 -0.08 FTCSR(SEQ ID NO: 86 and 87)

Example 11: Size Exclusion Chromatography and Affinity Measurements ofWild-Type and Mutant V_(H)Hs

Wild-type and mutant V_(H)Hs were passed over a SUPERDEX 75 (GEHealthcare) size exclusion chromatography column as described in Example7 to determine their aggregation state. Both wild-type and mutantV_(H)Hs were determined to be non-aggregating monomers (FIGS. 5, 17A).Similar size exclusion profiles were obtained for mutant and wild-type,indicating the second disulfide bond does not promote the formation ofinterdomain disulfide-bonds or multimeric mutant V_(H)Hs. The elutionvolumes (V_(e)s) of SEC molecular weight standards are shown with arrowsin FIG. 17A and are aligned relative to the A4.2 and A4.2mchromatograms. a: ovalbumin (MW=43.0 kDa, V_(e)=8.90 ml); b: carbonicanhydrase (MW=30.0 kDa, V_(e)=9.71 ml); c: typsin inhibitor (MW=20.1kDa, V_(e)=11.06 ml); d: α-lactalbumin (MW=14.4 kDa, V_(e)=11.97 ml); e:vitamin B (MW=1.3 kDa, V_(e)=18.7 ml). The equation of the line of astandard curve generated from these standards wasLOG₁₀MW=−0.1539V_(e)+2.9949 (r²=0.9995). From this equation the V_(H)Happarent MWs ranged from 9.8-13.6 kDa, indicating monomeric V_(H)Hs.

All kinetic rate and equilibrium constants of the mutants weredetermined as described (Example 7) using a BIACORE 3000 instrument fromGE Healthcare and 10,287 RUs of immobilized TcdA. SPR analysis revealedthe specific and high-affinity binding of 4 of 6 mutant V_(H)Hs to TcdA(FIG. 17B, Table 4). These four were also the strongest neutralizers(see Example 14). Two mutants (A19.2m and A24.1m) exhibited non-specificbinding to reference cell proteins and as a result specific interactiondata could not be generated, even at antibody concentrations as high as3.2 μM. When compared to their wild-type counterparts, the K_(D)s of 3TcdA-binding mutants were reduced approximately 2-6 fold (Table 4),while the affinity of one V_(H)H was relatively unchanged (K_(D)s of 24nM and 20 nM for A4.2 and A4.2m, respectively). The K_(D) reductionswere largely a result of faster k_(off) values and to a much lesserextent influenced by slower k_(on) values. Without wishing to be boundby theory, the Cys⁵⁴-Cys⁷⁸ disulfide bond may slightly distort theV_(H)H structure leading to decreases in target binding affinities anddecreases in antibody specificity.

TABLE 4 Kinetic and affinity constants of wild-type and mutant V_(H)Hsbinding to TcdA. Fold Wild-type Mutant change in V_(H)H k_(on) (M⁻¹s⁻¹)koff (s−1) K_(D) (nM) k_(on) (M⁻¹s⁻¹) koff (s−1) K_(D) (nM) K_(D) ^(a)A4.2/A4.2m 6.7 × 10⁵ 1.6 × 10⁻² 24 9.3 × 10⁵ 1.9 × 10⁻² 20 −1.2A5.1/A5.1m 1.6 × 10⁶ 5.0 × 10⁻³ 3 9.5 × 10⁵ 1.6 × 10⁻² 17 +5.7 A19.2/1.4 × 10⁴ 3.9 × 10⁻³ 290 — — — A19.2m A20.1/ 8.2 × 10⁵ 1.6 × 10⁻³ 2 6.4× 10⁵ 5.9 × 10⁻³ 9.2 +4.6 A20.1m A24.1/ 6.0 × 10⁴ 1.6 × 10⁻² 260 — — —A24.1m A26.8/ 1.4 × 10⁶ 1.6 × 10⁻² 12 1.0 × 10⁶ 2.8 × 10⁻² 28 +2.3A26.8m NB: no binding detected by BIACORE, at V_(H)H concentrations ashigh as 3.2 μM. ^(a)Relative to wild-type V_(H)H.

Example 12: Circular Dichroism Analysis of Mutant and Wild-Type V_(H)Hs

Circular dichroism (CD) experiments were used to examine V_(H)Hsecondary structure, tertiary structure, thermal refolding efficiency,and thermal stability at both neutral and acidic pH.

Wild-type and mutant V_(H)Hs were analyzed by circular dichroismspectrophotometry using a Jasco J-815 Spectrophotometer (Jasco, Easton,Md.) at pH 7.3 in 10 mM phosphate buffer (PB; 1.4 g/ml Na₂HPO₄+0.24 g/mlKH₂PO₄) and at pH 2.0 (10 mM PB+50 mM HCl). For all CD experimentsperformed at pH 2.0, proteins were equilibrated in the above buffer fora minimum of 2 h before scanning. For far-UV CD secondary structurescans, thermal refolding, and thermal unfolding experiments, a 5 mmcuvette containing 1.5 ml of V_(H)H at 50 μg/ml (3.2 μM; A₂₈₀≅0.1) wasused. In these experiments, data were collected for each sample between190-260 nm with a 1 mm bandwidth, 20 nm/min scan speed and 0.5 nm datapitch. Raw data was smoothed using the Jasco software, exported andconverted to mean residue ellipticity, [e]. Thermal unfolding wasfollowed at 215 nm by far-UV CD, with CD measurements taken every 2° C.from 30° C. to 96° C. with a temperature increase of 1° C./min. Meanresidue ellipticities [θ] were used to calculate the fraction of proteinfolded (FF) which is shown in Equation 1,

FF=([θ]−[θ_(U)])/([θ_(F)]−[θ_(U)])  Equation 1

where [θ_(F)] and [θ_(U)] are the molar ellipticities of the folded (30°C.) and unfolded (96° C.) states, respectively. The thermal unfoldingmidpoint temperature (T_(m)) was obtained by plotting FF againsttemperature and performing sigmoidal Boltzmann curve fitting in GraphPadPrism (GraphPad Software, Inc., La Jolla, Calif.). For refoldingexperiments, V_(H)Hs were first scanned (190 nm-260 nm) at 25° C.(folded); heated at 96° C. for 20 min and scanned (unfolded); andequilibrated to 25° C. for 3 h before a third scan (refolded). Raw datawas converted as before and thermal refolding efficiencies (TRE) werecalculated at 215 nm using Equation 2,

TRE=(([θ_(U)]−[θ_(R)])/([θ_(U)]−[θ_(F)]))×100  Equation 2

where [θ_(F)] is the molar ellipticity of the folded state acquired at25° C., [θ_(U)] is the molar ellipticity of the unfolded state acquiredat 96° C., and [θ_(R)] is the molar ellipticity of the refolded stateacquired at 25° C. To compare the tertiary structures of wild-type andmutant V_(H)Hs at neutral and acidic pH, near-UV CD experiments wereperformed in the range of 250 nm-340 nm using the conditions describedabove with the exception that a 10 mm cuvette containing 2 ml of proteinat 250 μg/ml was used. In all cases, the ellipticity of buffer blankswere subtracted from experimental values and the reported data is theaverage of two independent experiments with 4 data accumulations ineach.

Far-UV CD examined the V_(H)H secondary structure, and results are shownin FIG. 18. Although the overall shape of the far-UV CD spectra fromwild-type and mutant V_(H)H pairs was similar at a given pH, spectraintensity shifts were observed for all wild-type/mutant pairs. Ingeneral, peak minima were seen at 216 nm-218 nm and at 230 nm-235 nmwavelengths but, in almost all cases, the intensity of the peak at 216nm-218 nm was lower (decreased negative ellipticity) for mutant V_(H)Hs.Another prominent feature in the far-UV CD spectra was that mutantV_(H)Hs exhibited a near-UV shift in the peak range of 230 nm-235 nm.Wild-type V_(H)Hs possessed peak minima around 230 nm-232 nm whereasmutants displayed peak minima in this region around 232 nm-235 nm.Interestingly, A4.2/A4.2m, which had the most similar CD spectra atneutral pH of all the wild-type/mutant pairs, also had the same bindingaffinity for TcdA.

V_(H)H tertiary structures were analyzed with near-UV CD spectroscopy,and results are shown in FIG. 19. Overall, the near-UV spectra profileswere similar between wild-type and mutant V_(H)H pairs. Spectra fromwild-type and mutant pairs shared nearly identical peak wavelengths;however, between 250 nm to 295 nm, the ellipticity of mutant V_(H)Hs wasconsistently more negative than wild-type V_(H)Hs. There were alsosubtle differences in peaks occurring around 297 nm, with mutant V_(H)Hsexhibiting a minor but consistent shift to the right. Three of the fourwild-type/mutant pairs (A4.2/A4.2m, A5.1/A5.1m, and A20.1m/A20.1m)produced predominantly negative ellipticity, whereas the A26.8/A26.8mpair remained positive. The contributions of the second disulfide bondcannot be ruled out as a factor which may augment the contribution ofaromatic residues to ellipticity (increasing negatively) of the mutants.

Thermal refolding efficiencies (TREs) of wild-type and mutant V_(H)Hs atneutral and acid pH were also determined by far-UV CD. CD scans wereperformed at 50 μg/ml concentrations of V_(H)Hs (3.1 μM) at 25° C.(folded), after heating to 96° C. (unfolded), and after cooling for 3 hto 25° C. (refolded). Thermal refolding efficiencies were determined asthe extent to which the CD spectrum of the heated-and-cooled V_(H)Happroached that of the folded form. At pH 7.3, the TRE of wild-typeV_(H)Hs was essentially 100% and significantly higher than for themutants (FIG. 20, p=0.018, unpaired two-tailed t-test). Specifically,wild-type V_(H)Hs possessed a mean TRE of 99.7%±0.2% compared to mutantV_(H)Hs with a mean TRE of 90.0%±3.4% (FIG. 20). The ability of V_(H)Hsto refold in acidic conditions was also examined and, in general,mutants showed higher TREs at pH 2.0 (FIG. 20, Table 5). The mean TRE ofwild-type V_(H)Hs in acid was 68.2%±9.4% compared to 80.6%±4.8% formutant TREs in acid (FIG. 20). However, the mutant TREs were notsignificantly higher (p=0.268, unpaired two-tailed t-test). It should benoted that the TRE of 5 of 6 mutant V_(H)Hs increased in acidicconditions, with the TRE of A26.8m reaching 64.7% compared to only 25.8%for A26.8.

TABLE 5 Thermal refolding efficiencies (TREs) of wild-type and mutantV_(H)Hs at pH 2.0. V_(H)H TRE (%) TRE (%) A4.2 80.0 ± 3.8 87.4 ± 1.6A5.1 66.9 ± 4.2 87.2 ± 3.1 A19.2 78.3 ± 4.9 91.6 ± 1.4 A20.1 65.7 ± 2.585.9 ± 2.2 A24.1 92.7 ± 0.6 66.7 ± 0.3 A26.8  25.8 ± 17.6 64.7 ± 2.2 TRE(%) ± SEM (n = 8).

Temperature-induced unfolding experiments were conducted in order todetermine V_(H)H T_(m)s and T_(onset)s by following changes in V_(H)Hellipticity at 215 nm (FIG. 21; Tables 6, 7). All V_(H)Hs exhibitedsigmoidal melting curves. The wild-type V_(H)Hs have high T_(m)s (ashigh as 84.7° C.)—significantly higher than those reported for otherV_(H)Hs [60]. For all six V_(H)Hs, the mutants possessed higher T_(m)values, at both neutral and acidic pH (FIG. 21 and Table 6). At neutralpH, the T_(m) values of mutants ranged from 78.8° C. to 93.6° C., withone mutant, A5.1m, having a T_(m) 11.6° C. higher than wild-type (A5.1).The increase in mutant V_(H)H T_(m)s relative to wild-type ranged from3.7° C. to 11.6° C. Overall, at neutral pH, the mean T_(m)±SEM was 76.2°C.±1.8° C. and 83.6° C.±2.3° C. for wild-type and mutant V_(H)Hs,respectively (FIG. 21B). At acidic pH a considerable reduction in T_(m)was observed for both wild-type (22.1° C. to 32.4° C.) and mutantV_(H)Hs (23.7° C. to 31.2° C.) when compared to the T_(m) valuesrecorded at pH 7.3. However, at acidic pH the T_(m) of all six mutantswas still significantly higher than the corresponding wild-type V_(H)Hs(p=0.002, unpaired two-tailed t-test). In acid, the increase in mutantV_(H)H T_(m)s relative to wild-type ranged from 2.1° C. to 11.6° C.,which is a nearly identical spread in temperature increases to that seenat neutral pH. Overall, at pH 2.0, the mean T_(m)±SEM was 49.3° C.±1.2°C. and 56.6° C.±1.2° C. for wild-type and mutant V_(H)Hs, respectively(FIG. 21B). Interestingly, the highest T_(m) gains at both pH were seenfor the four strongest neutralizers. The T_(m) differences betweenwild-type/mutant pairs are more significant at acidic pH than neutralpH. Without wishing to be bound by theory, these results (Table 6; FIG.21) suggest the Cys⁵⁴-Cys⁷⁸ disulfide bond may stabilize the V_(H)Hsfrom acid-induced denaturation.

For example, A5.1 wild-type V_(H)H had a T_(m) of 73.1° C. and 45.6° C.at neutral and acidic pH, respectively, while the A5.1 mutant (A5.1m)had a T_(m) of 84.7° C. and 57.2° C. at neutral and acidic pH,respectively.

TABLE 6 Comparison of wild-type (WT) and mutant (Mut) V_(H)H thermalunfolding midpoint temperatures (T_(m)) at neutral and acidic pH. T_(m)(° C.) at pH 7.3 T_(m) (° C.) at pH 2.0 Wild- Wild- V_(H)H type MutantΔT_(m) type Mutant ΔT_(m) A4.2/  84.7* 93.6* 8.9 52.3 62.4 10.1 A4.2mA5.1/ 73.1 84.7* 11.6 45.6 57.2 11.6 A5.1m A19.2/ 75.1 78.8  3.7 53.055.1 2.1 A19.2m A20.1/ 72.4 79.1  6.7 46.6 55.4 8.8 A20.1m A24.1/ 74.680.1  5.5 49.4 54.6 5.2 A24.1m A26.8/ 77.2 85.3* 8.1 48.8 54.8 6.0A26.8m *Minimum estimated T_(m).

Using the thermal unfolding curves, V_(H)H T_(onset) temperatures werealso identified; this is the temperature at which 5% of the V_(H)H wasunfolded (FIG. 21C; Table 7). The T_(onset) of mutant V_(H)Hs wassignificantly higher than wild-type V_(H)Hs at both neutral and acidicpH (p=0.027 and p=0.006, respectively, unpaired two-tailed t-test). TheT_(onset) differences between wild-type/mutant pairs are moresignificant at acidic pH than neutral pH. At pH 7.3, the meanT_(onset)±SEM was 68.9° C.±1.8° C. and 74.9° C.±1.5° C. for wild-typeand mutant V_(H)Hs, respectively. At pH 2.0, the mean T_(onset)±SEM was41.2° C.±1.3° C. and 47.3° C.±1.3° C. for wild-type and mutant V_(H)Hs,respectively. Therefore, the lowest T_(onset) for the mutants was 45.0°C., whereas two of the wild-type V_(H)Hs (A5.1, A20.1) already hadT_(onset)s of ˜37° C. at pH 2.0 (physiological stomach conditions).

TABLE 7 Onset temperatures (T_(onset)s) of wild-type and mutant V_(H)Hs.T_(onset) pH 7.3 (° C.) T_(onset) pH 2.0 (° C.) V_(H)H Wild-type MutantWild-type Mutant A4.2/A4.2m 76.5 80.0 43.7 53.1 A5.1/A5.1m 65.2 76.637.8 48.4 A19.2/A19.2m 68.3 71.4 45.3 45.0 A20.1/A20.1m 64.6 72.0 37.846.3 A24.1/A24.1m 68.2 71.7 42.2 46.0 A26.8/A26.8m 70.7 77.8 40.3 45.2T_(onset) is defined as the temperature at which 5% of the V_(H)H isunfolded.

Example 13: Protease Resistance Profile Analysis of Mutant and Wild-TypeV_(H)Hs

The sensitivity of wild-type and mutant V_(H)Hs to the three majorgastrointestinal proteases pepsin, trypsin, and chymotrypsin, wasexplored to determine whether the Cys⁵⁴-Cys⁷⁸ disulfide bond improvedV_(H)H resistance to proteolytic degradation. The effects of theproteases were analyzed by SDS-PAGE and MS analysis.

All reactions were performed in 20 μl volumes with 4.8 μg of V_(H)Hdiluted in PBS pH 7.3. For pepsin digestions, reactions contained 17 μlof V_(H)H, 2 μl of porcine stomach pepsin (460 U/mg; Sigma), and 1 μl of1 M HCl (final pH=2.0). Final pepsin concentrations in each reactionranged from 0.1 μg/ml to 100 μg/ml. Digestions were incubated at 37° C.for 60 min and neutralized with 1 μl of 1 M NaOH. For trypsin andchymotrypsin digestions, reactions contained 18 μl of V_(H)H (diluted inPBS supplemented with 10 mM CaCl₂) and 2 μl of either trypsin orchymotrypsin (sequencing grade, Hoffmann-La Roche). Finaltrypsin/chymotrypsin concentrations ranged from 0.1 μg/ml to 100 μg/ml.Digestions were incubated at 37° C. for 60 min and neutralized with 1 μlof protease inhibitor cocktail (Sigma). All neutralized V_(H)H-proteasereactions and controls (V_(H)Hs with no protease) were separated bySDS-PAGE, stained with Coomassie and photographed using an ALPHAIMAGER3400 (Alpha Innotech Corporation, San Leandro, Calif.). To determine thepercent of V_(H)H retained after protease digestions, densitometryanalysis was performed using the ALPHAEASE Fc software package (Version7.0.1, Alpha Innotech Corporation) on control and digested V_(H)Hs. Atotal of three independent digestion reactions were performed on all ofthe V_(H)Hs at each protease concentration and each were run on separateSDS-PAGE gels. Digestions at the highest protease concentration (100μg/ml) that were not analyzed by SDS-PAGE were buffer exchanged intoddH₂O using Millipore BIOMAX 5K MWCO spin columns (Millipore, Billerica,Mass.) and subjected to MS analysis to identify the cleavage products,or analyzed by SPR for TcdA binding activity.

Protease concentrations of 0.1 μg/ml, 1 μg/ml, 10 μg/ml, and 100 μg/mlwere explored. When the lowest concentrations of proteases (0.1 μg/mland 1 μg/ml) were used in digestion reactions, wild-type and mutantsappeared similar to undigested controls on SDS-PAGE (data not shown).Similarly, V_(H)Hs were only moderately susceptible to proteasedegradation at 10 μg/ml (data not shown). In order to see cleardifferences in the proteolytic susceptibility of wild-type and mutantV_(H)Hs, all remaining digestions were performed at proteaseconcentrations of 100 μg/ml.

A representative SDS-PAGE gel comparing A5.1 wild-type and mutant V_(H)Hdigestion with various concentrations of pepsin is shown in FIG. 22A. Areduction in V_(H)H size from ˜16 kDa (control) to either ˜14 kDa, orcomplete digestion to smaller fragments can be observed. The band at ˜14kDa routinely appeared in digestions with each of the proteases and wasshown by MS mass analysis to correspond to cleavage at various positionswithin the V_(H)H C-terminal c-Myc epitope tag. Loss of the epitope tagcorresponded to reductions of 1641.7 Da, 1754.8 Da, and 1641.7 Da forpepsin, trypsin, and chymotrypsin digested V_(H)Hs, respectively (datanot shown). Overall, significant increases in pepsin resistance werefound for all mutant V_(H)Hs compared to their wild-type counterparts(p=0.026, Mann-Whitney U test) (FIG. 22B, E; Table 8). All mutantV_(H)Hs were found to possess greater pepsin resistance, a protease thatfunctions at acidic pH, compared to the wild-type V_(H)Hs (p=0.026,Mann-Whitney U test) (FIG. 22B, E and Table 8). The increase in mutantV_(H)H pepsin resistance relative to corresponding wild-type ranged fromalmost 4.5% to 63% (Table 8). For example, A5.1 was completely degradedafter incubation with pepsin, while nearly 50% of A5.1m remained intact(FIG. 22A, B). The biggest increase in pepsin resistance was found forA4.2m, where an almost 63% increase in intact V_(H)H structure was foundrelative to A4.2. Interestingly, A4.2m also had the highest T_(m) andT_(onset) at pH 2.0 (Table 7), the same pH at which the pepsindigestions were performed.

In general, V_(H)Hs with a higher T_(m) at pH 2 correlated with agreater resistance to pepsin degradation (FIG. 23A) (R²=0.735). Thus,while wild type V_(H)Hs with lower T_(m)s occupied the low proteaseresistance region of the graph, the mutants with higher T_(m)s occupiedthe high protease resistance region of the graph. There was also amoderate correlation between V_(H)H pepsin resistance and T_(m)s at pH7.3 (r²=0.500, data not shown). In addition, a strong correlationbetween wild-type V_(H)H pepsin resistance and wild-type V_(H)HT_(onset) at pH 2.0 was noted (r²=0.975, FIG. 23B). No correlation wasevident between mutant V_(H)H pepsin resistance and mutant V_(H)HT_(onset) at pH 2.0 (r²=0.191, data not shown); without wishing to bebound by theory, this was likely because mutant V_(H)H T_(onset)temperatures were much higher than the temperature at which pepsindigestions were performed (37° C.).

TABLE 8 Comparison of wild-type (WT) and mutant (Mut) V_(H)H resistanceprofiles to the major gastrointestinal proteases. All V_(H)H digestionswere performed at 37° C. for 1 hour in the presence of 100 μg/ml ofprotease; resistance profiles were obtained by comparing the intensityof protease- digested V_(H)Hs relative to untreated controls usingSDS-PAGE and imaging software; values represent the mean ± SEM of threeindependent experiments. Chymotrypsin Resistance Pepsin Resistance (%)Trypsin Resistance (%) (%) V_(H)H WT Mut WT Mut WT Mut A4.2 11.08 ± 1.8873.87 ± 7.23 35.72 ± 7.08  4.80 ± 0.61 13.60 ± 6.50  3.18 ± 1.10 A5.1 0.53 ± 0.15 46.63 ± 1.99 96.23 ± 7.09 83.30 ± 4.96 14.03 ± 3.15 27.00 ±4.05 A19.2 30.37 ± 3.16 52.27 ± 0.32  0.73 ± 0.73  0.27 ± 0.27  8.30 ±1.14  0.18 ± 0.10 A20.1  0.68 ± 0.68  5.04 ± 0.76 72.77 ± 4.85 82.80 ±1.97 10.17 ± 1.85 16.17 ± 5.26 A24.1 10.45 ± 2.39 36.02 ± 1.11 75.03 ±9.63 66.50 ± 3.58 22.03 ± 5.01 43.80 ± 2.08 A26.8  3.17 ± 1.24 24.56 ±1.45  2.03 ± 2.03  4.10 ± 1.27  8.40 ± 1.23 40.83 ± 8.81

The resistance profiles of wild-type and mutant V_(H)Hs to trypsin andchymotrypsin, proteases that function at neutral pH, were more variedwithout a clearly defined trend (FIG. 22C-E and Table 8). 4 of 6 mutantV_(H)Hs showed increased resistance to chymotrypsin, with significantincreases found in clones A5.1m, A24.1m, and A26.8m (p<0.05) compared totheir wild-type counterparts. No statistical differences were foundbetween trypsin digested wild-type and mutant V_(H)Hs (FIG. 22C-E; Table8), except for A4.2m, where trypsin resistance was actually reduced fromalmost 36% in the wild-type V_(H)H to almost 5% in the mutant. Both thewild-type and mutant versions of A19.2 and A26.8 were very susceptibleto trypsin degradation. No correlation was evident between V_(H)Htrypsin resistance and T_(m)s at pH 7.3 or pH 2.0 (r²=0.138 andr²=0.138, respectively) or between V_(H)H chymotrypsin resistance andT_(m)s at pH 7.3 or pH 2.0 (r²=0.012 and r²=0.004, respectively).

The ability of pepsin-treated mutants (A4.2m, A5.1m, A20.1m, and A26.8m)to bind TcdA was evaluated by SPR. SPR analyses confirmed thepepsin-treated mutants (“V_(H)H-tag”) retained TcdA binding as theirk_(off) values (FIG. 23C) were essentially the same as those ofuntreated controls (Table 4; FIG. 23C). SPR analysis on pepsin-digestedwild-type V_(H)Hs could not be performed since these V_(H)Hs weresignificantly degraded by pepsin. Without wishing to be bound by theory,this highlights the impact a second disulfide bond in the hydrophobiccore has on V_(H)H conformational stability at low pH and resistance toproteolytic degradation by pepsin.

Example 14: TcdA Toxin Neutralization Assay

In vitro TcdA neutralization assays were performed essentially asdescribed [20]. Human lung fibroblast cell rounding was reported 24 hpost addition of TcdA (100 ng/ml), TcdA+wild-type V_(H)H (1000 nM) orTcdA+mutant V_(H)H (1000 nM). Specifically, V_(H)Hs were added as pooledmixtures of A4.2, A5.1, A20.1, and A26.8 (250 nM each, 1000 nM total) orA4.2m, A5.1m, A20.1m, and A26.8m (250 nM each, 1000 nM total). Thepercentage of cell rounding was scored visually using light microscopyand the reported values are the average of two independent experimentsin which each V_(H)H mixture was tested in triplicate.

Mutant V_(H)Hs retained their ability to neutralize to cytotoxic effectsof TcdA on monolayers of fibroblast cells. Comparison of theneutralization capacity of pooled mixtures (1000 nM total) of wild-typeand mutant V_(H)Hs revealed mutants performed nearly as well aswild-types at reducing TcdA-mediated cell rounding (FIG. 24). Given that3 of 4 mutants showed weaker affinity for TcdA the reduction inneutralizing capacity relative to wild-type V_(H)Hs was not unexpected.

As indicated above, the mutant antibodies were compared to theirwild-type counterparts with respect to expression, yield, solubility,affinity for TcdA, thermal unfolding at neutral and acidic pH, andprotease resistance. Mutant V_(H)Hs were found to be soluble,non-aggregating monomers, possessing similar affinity constants to thatof WT V_(H)Hs. A significant increase in the midpoint temperature ofunfolding (4-12° C.) was observed for all mutants, at both neutral andacidic pH (p<0.05; unpaired two-tailed t test). Digestion of the V_(H)Hswith major gastrointestinal proteases at biologically relevantconcentrations revealed a significant increase in pepsin resistance forall mutants (p<0.05; unpaired two-tailed Mann-Whitney U test), However,increases in resistance profiles to chymotrypsin and trypsin were not asuniversal. Overall, the introduction of an additional disulfide bond inthe hydrophobic core of the anti-TcdA V_(H)Hs not only increased thermalstability at neutral pH, but also represents a generic strategy toincrease antibody stability at low pH and impart pepsin resistance whichis desirable for protein-based oral therapeutics.

Example 15: Sequence Identities Between V_(H)Hs

Sequence identities between the toxin A binders, as well as toxin Bbinders were determined.

The sequences of V_(H)H pairs were aligned using ClustalW (Thompson etal, 1994), and the percentage identity between the V_(H)H pairs wascalculated using the BIOEDIT Sequence Alignment Editor. Results areshown in Tables 9 and 10, below.

TABLE 9 Percentage amino acid sequence identities between TcdA-bindingV_(H)Hs. V_(H)H 1 V_(H)H 2 Identity (%) A4.2 A5.1 82 A4.2 A19.2 76 A4.2A20.1 82 A4.2 A24.1 78 A4.2 A26.8 75 A5.1 A19.2 77 A5.1 A20.1 78 A5.1A24.1 77 A5.1 A26.8 78 A19.2 A20.1 77 A19.2 A24.1 75 A19.2 A26.8 75A20.1 A24.1 80 A20.1 A26.8 77 A24.1 A26.8 74 A4.2 A4.2m 98.4 A5.1 A5.1m98.4 A19.2 A19.2m 98.4 A20.1 A20.1m 98.4 A24.1 A24.1m 98.4 A26.8 A26.8m98.4

TABLE 10 Percentage amino acid sequence identities between TcdB-bindingV_(H)Hs. V_(H)H 1 V_(H)H 2 Identity (%) B5.2 B7.3 69 B5.2 B13.6 81 B5.2B15.3 66 B5.2 B15.5 73 B7.3 B13.6 74 B7.3 B15.3 72 B7.3 B15.5 69 B13.6B15.3 68 B13.6 B15.5 71 B15.3 B15.5 69 B5.2 B5.2m 98.3 B7.3 B7.3m 98.4B13.6 B13.6m 98.3 B15.3 B15.3m 98.4 B15.5 B15.5m 98.3

The embodiments and examples described herein are illustrative and arenot meant to limit the scope of the invention as claimed. Variations ofthe foregoing embodiments, including alternatives, modifications andequivalents, are intended by the inventors to be encompassed by theclaims. Furthermore, the discussed combination of features might not benecessary for the inventive solution.

REFERENCES

All patents, patent applications and publications referred to herein andthroughout the application are hereby incorporated by reference.

-   Anderson, G. P., Liu, J. L., Hale, M. L., Bernstein, R. D., Moore,    M., Swain, M. D., and Goldman, E. R. (2008) Anal. Chem. 80,    9604-9611.-   Arbabi-Ghahroudi, M., MacKenzie, R., and Tanha, J. (2009c) Methods    Mol. Biol. 525, 187-216.-   Arbabi-Ghahroudi, M., MacKenzie, R., and Tanha, J. (2010) Methods    Mol. Biol. 634, 309-330.-   Arbabi-Ghahroudi, M., To, R., Gaudette, N., Hirama, T., Ding, W.,    MacKenzie, R., and Tanha, J. (2009b) Protein Eng. Des. Sel. 22,    59-66.-   Babcock, G. J., Broering, T. J., Hernandez, H. J., Mandell, R. B.,    Donahue, K., Boatright, N., Stack, A. M., Lowy, I., Graziano, R.,    Molrine, D., Ambrosino, D. M., and Thomas, W. D. Jr. (2006) Infect.    Immun. 74, 6339-6347.-   Bell, A., Wang, Z. J., Arbabi-Ghahroudi, M., Chang, T. A., Durocher,    Y., Trojahn, U., Baardsnes, J., Jaramillo, M. L., Li, S., Baral, T.    N., O'Connor-McCourt, M., Mackenzie, R., and Zhang, J. (2010) Cancer    Lett. 289, 81-90.-   Chothia, C., and Lesk, A. M. (1987) J. Mol. Biol. 196, 901-917.-   Corthier, G., Muller, M. C., Wilkins, T. D., Lyerly, D., and    L'Haridon, R. (1991) Infect. Immun. 59, 1192-1195.-   De Genst, E., Silence, K., Decanniere, K., Conrath, K., Loris, R.,    Kinne, J., Muyldermans, S., and Wyns, L. (2006) Proc. Natl. Acad.    Sci. U.S.A. 103, 4586-4591.-   De Kruif, J., and Logtenberg, T. (1996) J. Biol. Chem. 271,    7630-7634.-   Doyle, P. J., Arbabi-Ghahroudi, M., Gaudette, N., Furzer, G.,    Savard, M. E., Gleddie, S., McLean, M. D., MacKenzie, C. R., and    Hall, J. C. (2008) Mol. Immunol. 45, 3703-3713.-   Eisenberg, D., Schwarz, E., Komaromy, M., and Wall, R. (1984) J.    Mol. Biol. 179, 125-142-   Fenner, L., Widmer, A. F., Goy, G., Rudin, S., and    Frei, R. (2008) J. Clin. Microbiol. 46, 328-330.-   Florin, I., and Thelestam, M. (1983) Biochim. Biophys. Acta 763,    383-392.-   Gardiner, D. F., Rosenberg, T., Zaharatos, J., Franco, D., and    Ho, D. D. (2009) Vaccine 27, 3598-3604.-   Giannasca, P. J., Zhang, Z. X., Lei, W. D., Boden, J. A., Giel, M.    A., Monath, T. P., and Thomas, W. D. Jr. (1999) Infect. Immun. 67,    527-538.-   Goldman, E. R., Anderson, G. P., Conway, J., Sherwood, L. J., Fech,    M., Vo, B., Liu, J. L., and Hayhurst, A. (2008) Anal. Chem. 80,    8583-8591.-   Goldman, E. R., Anderson, G. P., Liu, J. L., Delehanty, J. B.,    Sherwood, L. J., Osborn, L. E., Cummins, L. B., and    Hayhurst, A. (2006) Anal. Chem. 78, 8245-8255.-   Greco, A., Ho, J. G., Lin, S. J., Palcic, M. M., Rupnik, M., and    Ng, K. K. (2006) Nat. Struct. Mol. Biol. 13, 460-461.-   Hagihara, Y., Mine, S., and Uegaki, K. (2007) J. Biol. Chem. 282,    36489-36495.-   Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G.,    Hamers, C., Songa, E. B., Bendahman, N., and Hamers, R. (1993)    Nature 363, 446-448.-   Hassoun, A., and Ibrahim, F. (2007) Am. J. Geriatr. Pharmacother. 5,    48-51.-   Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and    Pease, L. R. (1989) Gene 77, 51-59.-   Hmila, I., Abdallah, R. B. A., Saerens, D., Benlasfar, Z., Conrath,    K., Ayeb, M. E., Muyldermans, S., and Bouhaouala-Zahar, B. (2008)    Mol. Immunol. 45, 3847-3856.-   Hussack, G., Luo, Y., Veldhuis, L., Hall, J. C., Tanha, J., and    MacKenzie, R. (2009) Sensors 9, 5351-5367.-   Hussack, G., Hirama, T., Ding, W., MacKenzie, R., and    Tanha, J. (2011) PLoS ONE, In press.-   Iqbal, U., Trojahn, U., Albaghdadi, H., Zhang, J., O'Connor, M.,    Stanimirovic, D., Tomanek, B., Sutherland, G., and    Abulrob, A. (2010) Br. J. Pharmacol. 160, 1016-1028.-   Jank, T., and Aktories, K. (2008) Trends Microbiol. 16, 222-229.-   Jank, T., Giesemann, T., and Aktories, K. (2007) Glycobiology 17,    15R-22R.-   Jespers, L., Schon, O., Famm, K., and Winter, G. (2004) Nat.    Biotechnol. 22, 1161-1165.-   Johal, S. S., Lambert, C. P., Hammond, J., James, P. D.,    Borriello, S. P., and Mahida, Y. R. (2004) J. Clin. Pathol. 57,    973-979.-   Johnson, S. (2009) J. Infect. 58, 403-410.-   Juang, P., Skledar, S. J., Zgheib, N. K., Paterson, D. L.,    Vergis, E. N., Shannon, W. D., Ansani, N. T., and    Branch, R. A. (2007) Am. J. Infect. Control 35, 131-137.-   Kabat, E. A., and Wu, T. T. (1991) J. Immunol. 147:1709-19.-   Katchar, K., Taylor, C. P., Tummala, S., Chen, X., Sheikh, J., and    Kelly, C. P. (2007) Clin. Gastroenterol. Hepatol. 5, 707-713.-   Keel, M. K., and Songer, J. G. (2007) Vet. Pathol. 44, 814-822.-   Kelly, C. P., Chetham, S., Keates, S., Bostwick, E. F., Roush, A.    M., Castagliuolo, I., LaMont J. T., and Pothoulakis, C. (1997)    Antimicrob. Agents Chemother. 41, 236-241.-   Kelly, C. P., Pothoulakis, C., and LaMount, J. T. (1994) N. Engl. J.    Med. 330, 257-262.-   Kelly, C. P., Pothoulakis, C., Orellana, J., and    LaMont, J. T. (1992) Gastroenterology 102, 35-40 (2009) Nat. Rev.    Drug Discov. 8, 442.-   Kelly, C. P., Pothoulakis, C., Vavva, F., Castagliuolo, I.,    Bostwick, E. F., O'Keane, J. C., Keates, S., and    LaMont, J. T. (1996) Antimicrob. Agents Chemother. 40, 373-379.-   Kink, J. A., and Williams, J. A. (1998) Infect. Immun. 66,    2018-2025.-   Kyne, L., Hamel, M. B., Polavaram, R., and Kelly, C. P. (2002) Clin.    Infect. Dis. 34, 346-353.-   Kyne, L., Warny, M., Qamar, A., and Kelly, C. P. (2000) N. Engl. J.    Med. 340, 390-397.-   Kyne, L., Warny, M., Qamar, A., and Kelly, C. P. (2001) Lancet 357,    189-193.-   Leffler, D. A., and Lamont, J. T. (2009) Gastroenterology 136,    1899-1912.-   Lefranc, M.-P. et al., (2003) Dev. Comp. Immunol., 27, 55-77.-   Leung, D. Y., Kelly, C. P., Boguniewicz, M., Pothoulakis, C.,    LaMont, J. T., and Flores, A. (1991) J. Pediatr. 118, 633-637.-   Liu, J. L., Anderson, G. P., and Goldman, E. R. (2007a) BMC    Biotechnol. 7, 78-88.-   Lowy, I., Molrine, D. C., Leav, B. A., Blair, B. M., Baxter, R.,    Gerding, D. N., Nichol, G., Thomas, W. D. Jr., Leney, M., Sloan, S.,    Hay, C. A., and Ambrosino, D. M. (2010) N. Engl. J. Med. 362,    197-205.-   Lyerly, D. M., Bostwick, E. F., Binion, S. B., and    Wilkins, T. D. (1991) Infect. Immun. 59, 2215-2218.-   Lyerly, D. M., Phelps, C. J., Toth, J., and Wilkins, J. D. (1986)    Infect. Immun. 54, 70-76.-   Lyras, D., O'Connor, J. R., Howarth, P. M., Sambol, S. P.,    Carter, G. P., Phumoonna, T., Poon, R., Adams, V., Vedantam, G.,    Johnson, S., Gerding, D. N., and Rood, J. I. (2009) Nature 458,    1176-1179.-   McPherson, S., Rees, C. J., Ellis, R., Soo, S., and    Panter, S. J. (2006) Dis. Colon Rectum 49, 640-645.-   Merritt, E. A., and Hol, W. G. (1995) Curr. Opin. Struct. Biol. 5,    165-171.-   Musher, D. M., Manhas, A., Jain, P., Nuila, F., Waqar, A., Logan,    N., Marino, B., Graviss, E. A. (2007) J. Clin. Microbiol. 45,    2737-2739.-   Nielsen, U. B., Adams, G. P., Weiner, L. M., and Marks, J. D. (2000)    Cancer Res. 60, 6434-6440.-   Nuttall, S. D., Krishnan, U. V., Doughty, L., Pearson, K., Ryan, M.    T., Hoogenraad, N. J., Hattarki, M., Carmichael, J. A., Irving, R.    A., and Hudson, P. J. (2003) Eur. J. Biochem. 270, 3543-3554.-   O'Connor, J. R., Johnson, S., and Gerding, D. N. (2009)    Gastroenterology 136, 1913-1924.-   Ochsner, U. A., Bell, S. J., O'Leary, A. L., Hoang, T., Stone, K.    C., Young, C. L., Critchley, I. A., and Janjic, N. (2009) J.    Antimicrob. Chemother. 63, 964-971.-   Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995)    Protein Sci. 4, 2411-2423.-   Pépin, J., Valiquette, L., and Cossette, B. (2005) CMAJ 173,    1037-1042.-   Planche, T., Aghaizu, A., Holliman, R., Riley, P., Poloniecki, J.,    Breathnach, A., and Krishna, S. (2008) Lancet Infect. Dis. 8,    777-784.-   Ridgway, J. B., Presta, L. G., and Carter, P. (1996) Protein Eng. 9,    617-621.-   Rupnik, M., Wilcox, M. H., and Gerding, D. N. (2009) Nat. Rev.    Microbiol. 7, 526-536.-   Rüssmann, H., Panthel, K., Bader, R. C., Schmitt, C., and    Schaumann, R. (2007) Eur. J. Clin. Microbiol. Infect. Dis. 26,    115-119.-   Saerens, D., Conrath, K., Govaert, J., and Muyldermans, S. (2008) J.    Mol. Biol. 377, 478-488.-   Salcedo, J., Keates, S., Pothoulakis, C., Warny, M., Castagliuolo,    I., LaMont, J. T., and Kelly, C. P. (1997) Gut 41, 366-370.-   Salnikova, M. S., Joshi, S. B., Rytting, J. H., Warny, M., and    Middaugh, C. R. (2008) J. Pharm. Sci. 97, 3735-3752.-   Sloan, L. M., Duresko, B. J., Gustafson, D. R., and    Rosenblatt, J. E. (2008) J. Clin. Microbiol. 46, 1996-2001.-   Songer, J. G. (2004) Anim. Health Res. Rev. 5, 321-326.-   Stewart, C. S., MacKenzie, C. R., and Hall, J. C. (2007) Toxicon 49,    699-709.-   Tanha, J., Muruganandam, A., and Stanimirovic, D. (2003) Methods    Mol. Med. 89, 435-449.-   Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic    Acids Res. 22, 4673-4680.-   Tjellström, B., Stenhammar, L., Eriksson, S., and    Magnusson, K. E. (1993) Lancet 341, 701-702.-   To, R., Hirama, T., Arbabi-Ghahroudi, M., MacKenzie, R., Wang, P.,    Xu, P., Ni, F., and Tanha, J. (2005) J. Biol. Chem. 280,    41395-41403.-   Turgeon, D. K., Novicki, T. J., Quick, J., Carlson, L., Miller, P.,    Ulness, B., Cent, A., Ashley, R., Larson, A., Coyle, M., Limaye, A.    P., Cookson, B. T., and Fritsche, T. R. (2003) J. Clin. Microbiol.    41, 667-670.-   Viscidi, R., Laughon, B. E., Yolken, R., Bo-Linn, P., Moench, T.,    Ryeder, R. W., and Bartlett, J. G. (1983) J. Infect. Dis. 148,    93-100.-   Vu, K. B., Ghahroudi, M. A., Wyns, L., and Muyldermans, S. (1997)    Mol. Immunol. 34, 1121-1131.-   Warny, M., Fatimi, A., Bostwick, E. F., Laine, D. C., Label, F.,    LaMont, J. T., Pothoulakis, C., and Kelly, C. P. (1999) Gut 44,    212-217.-   Warny, M., Pepin, J., Fang, A., Killgore, G., Thompson, A., Brazier,    J., Frost, E., and McDonald, L. C. (2005) Lancet 366, 1079-1084.-   Warny, M., Vaerman, J. P., Avesani, V., and Delmee, M. (1994)    Infect. Immun. 62, 384-389.-   Wesolowski, J., Alzogaray, V., Reyelt, J., Unger, M., Juarez, K.,    Urrutia, M., Cauerhff, A., Danguah, W., Rissiek, B., Scheuplein, F.,    Schwarz, N., Adriouch, S., Boyer, O., Seman, M., Licea, A.,    Serreze, D. V., Goldbaum, F. A., Haag, F., and Koch-Nolte, F. (2009)    Med. Microbiol. Immunol. 198, 157-174.-   Wilcox, N. H. (2004) J. Antimicrob. Chemother. 53, 882-884.-   Wilkins, T. D., and Lyerly, D. M. (2003) J. Clin. Microbiol. 41,    531-534.-   Zhang, J., Li, Q., Nguyen, T.-D., Tremblay, T.-L., Stone, E., To,    R., Kelly, J., and MacKenzie, C. R. (2004a) J. Mol. Biol. 341,    161-169.-   Zhang, J., Tanha, J., Hirama, T., Khiew, N. H., To, R., Tong-Sevinc,    H., Stone, E., Brisson, J. R., and MacKenzie, C. R. (2004b) J. Mol.    Biol. 335, 49-56.

1. An isolated or purified antibody or fragment thereof, comprising asequence of complementarity determining region (CDR) 1 selected fromGRTFNTLS (SEQ ID NO:1); GRTFSMYR (SEQ ID NO:2); GRTLSSYI (SEQ ID NO:3);GRTFSMDP (SEQ ID NO:4); IRSFSNRN (SEQ ID NO:5); and ERTFSRYP (SEQ IDNO:6); a sequence of CDR2 selected from VSRSGGST (SEQ ID NO:7); ITRNGSST(SEQ ID NO:8); ISRRGGNS (SEQ ID NO:9); GSSTGRTT (SEQ ID NO:10); ISWGGGST(SEQ ID NO:11); and ISSTGTST (SEQ ID NO:12); and a sequence of CDR3selected from AAAATKSNTTAYRLSFDY (SEQ ID NO:13); AATSGSSYLDAAHVYDY (SEQID NO:14); AADGSVAGWGRRSVSVSSYDY (SEQ ID NO:15); AAAPYGANWYRDEYAY (SEQID NO:16); AAEFGHNIATSSDEYDY (SEQ ID NO:17); and AVNSQRTRLQDPNEYDY (SEQID NO:18), wherein the antibody or fragment thereof is specific forTcdA.
 2. The isolated or purified antibody or fragment thereof of claim1, selected from the group consisting of: an isolated or purifiedantibody or fragment thereof comprising a sequence of CDR 1 of GRTFNTLS(SEQ ID NO:1); CDR2 of VSRSGGST (SEQ ID NO:7); and CDR3 ofAAAATKSNTTAYRLSFDY (SEQ ID NO:13); an isolated or purified antibody orfragment thereof comprising a sequence of CDR 1 of GRTFSMYR (SEQ IDNO:2); CDR2 of ITRNGSST (SEQ ID NO:8); and CDR3 of AATSGSSYLDAAHVYDY(SEQ ID NO:14); an isolated or purified antibody or fragment thereofcomprising a sequence of CDR 1 of GRTLSSYI (SEQ ID NO:3); CDR2 ofISRRGGNS (SEQ ID NO:9); and CDR3 of AADGSVAGWGRRSVSVSSYDY (SEQ IDNO:15); an isolated or purified antibody or fragment thereof comprisinga sequence of CDR 1 of GRTFSMDP (SEQ ID NO:4); CDR2 of GSSTGRTT (SEQ IDNO:10); and CDR3 of AAAPYGANWYRDEYAY (SEQ ID NO:16); an isolated orpurified antibody or fragment thereof comprising a sequence of CDR 1 ofIRSFSNRN (SEQ ID NO:5); CDR2 of ISWGGGST (SEQ ID NO:11); and CDR3 ofAAEFGHNIATSSDEYDY (SEQ ID NO:17); or an isolated or purified antibody orfragment thereof comprising a sequence of CDR 1 of ERTFSRYP (SEQ IDNO:6); CDR2 of ISSTGTST (SEQ ID NO:12); and CDR3 of AVNSQRTRLQDPNEYDY(SEQ ID NO:18).
 3. The isolated or purified antibody or fragment thereofof claim 1, comprising a sequence selected from the group consisting of:(SEQ ID NO: 34) QVKLEESGGGLVQAGGSLRLSCAASGRTFNTLSMGWFRQAPGKEREFVAAVSRSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAAATKSNTTAYRLSFDYWGQGTQVTVSS; (SEQ ID NO: 35)QVKLEESGGGLVQAGGSLRLSCAASGRTFSMYRMGWFRQAPGKEREFVGVITRNGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATSGSSYLDAAHVYDYWGQGTQVTVSS; (SEQ ID NO: 36)QVKLEESGGGLVQPGGSLRLSCAASGRTLSSYIVAWFRQAPGKEREFVAGISRRGGNSAYVESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADGSVAGWGRRSVSVSSYDYWGQGTQVTVSS; (SEQ ID NO: 37)QVQLVESGGGLAQAGGSLRLSCAASGRTFSMDPMAWFRQPPGKEREFVAAGSSTGRTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAAPYGANWYRDEYAYWGQGTQVTVSS; (SEQ ID NO: 38)QVQLVESGGGLVQAGGSLRLSCAASIRSFSNRNMGWFRQPPGKEREFVAGISWGGGSTRYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEFGHNIATSSDEYDYWGQGTQVTVSS; (SEQ ID NO: 39)QVKLEESGGGLVQAGGSLRLSCAASERTFSRYPVAWFRQAPGAEREFVAVISSTGTSTYYADSVKGRFTISRDNAKVTVYLQMNNLKREDTAVYFCAVNSQRTRLQDPNEYDYWGQGTQVTVSS; (SEQ ID NO: 45)QVKLEESGGGLVQAGGSLRLSCAASGRTFNTLSMGWFRQAPGKEREFVCAVSRSGGSTYYADSVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVYYCAAAATKSNTTAYRLSFDYWGQGTQVTVSS; (SEQ ID NO: 46)QVKLEESGGGLVQAGGSLRLSCAASGRTFSMYRMGWFRQAPGKEREFVCVITRNGSSTYYADSVKGRFTCSRDNAKNTVYLQMNSLKPEDTALYYCAATSGSSYLDAAHVYDYWGQGTQVTVSS; (SEQ ID NO: 47)QVKLEESGGGLVQPGGSLRLSCAASGRTLSSYIVAWFRQAPGKEREFVCGISRRGGNSAYVESVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVYYCAADGSVAGWGRRSVSVSSYDYWGQGTQVTVSS; (SEQ ID NO: 48)QVQLVESGGGLAQAGGSLRLSCAASGRTFSMDPMAWFRQPPGKEREFVCAGSSTGRTTYYADSVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVYYCAAAPYGANWYRDEYAYWGQGTQVTVSS; (SEQ ID NO: 49)QVQLVESGGGLVQAGGSLRLSCAASIRSFSNRNMGWFRQPPGKEREFVCGISWGGGSTRYADSVKGRFTCSRDNAKKTVYLQMNSLKPEDTAVYYCAAEFGHNIATSSDEYDYWGQGTQVTVSS; and (SEQ ID NO: 50)QVKLEESGGGLVQAGGSLRLSCAASERTFSRYPVAWFRQAPGAEREFVCVISSTGTSTYYADSVKGRFTCSRDNAKVTVYLQMNNLKREDTAVYFCAVNSQRTRLQDPNEYDYWGQGTQVTVSS,

or a sequence substantially identical thereto.
 4. The isolated orpurified antibody or fragment thereof of any one of claims 1 to 3,wherein the antibody or fragment thereof binds to a conformational orlinear epitope.
 5. The isolated or purified antibody or fragment thereofof claim 1, wherein the antibody or fragment thereof is in a multivalentdisplay format.
 6. A nucleic acid molecule encoding the isolated orpurified antibody or fragment thereof of claim
 1. 7. A vector comprisingthe nucleic acid molecule of claim
 6. 8. The isolated or purifiedantibody or fragment thereof of claim 1, wherein the antibody orfragment thereof is immobilized onto a surface.
 9. The isolated orpurified antibody or fragment thereof of claim 1, wherein the antibodyor fragment thereof is linked to a cargo molecule.
 10. The isolated orpurified antibody or fragment thereof of claim 9, wherein the cargomolecule is a detectable agent, a therapeutic, a drug, a peptide, acarbohydrate moiety, an enzyme, or a cytotoxic agent; one or moreliposomes loaded with a detectable agent, a therapeutic, a drug, apeptide, an enzyme, or a cytotoxic agent; or one or more nanoparticle,nanowire, nanotube, or quantum dots.
 11. A composition comprising one ormore than one isolated or purified antibody or fragment thereof of claim1 and a pharmaceutically-acceptable carrier, diluent, or excipient. 12.A method of treating a Clostridium difficile infection, comprisingadministering the isolated or purified antibody or fragment thereof ofclaim 1 to a subject in need thereof.
 13. A method of capturingClostridium difficile toxins, comprising contacting a sample with one ormore than one isolated or purified antibody or fragment thereof of claim8 and allowing the toxin(s) to bind to the isolated or purified antibodyor fragment thereof.
 14. The method of claim 13, further comprisingidentification of the toxin by a mass spectrometric method.
 15. Themethod of claim 13, further comprising eluting the bound toxin.
 16. Themethod of claim 15, wherein elution of the bound toxin is accomplishedby heat elution.
 17. A method of detecting Clostridium difficile toxins,comprising contacting a sample with one or more than one isolated orpurified antibody or fragment thereof of claim 9 or 10, and detectingthe bound antibody or fragment thereof using a suitable detection and/orimaging technology.
 18. A method of treating a Clostridium difficileinfection, comprising administering isolated or purified single domainantibody, comprising a sequence selected from the group consisting of:(SEQ ID NO: 34) QVKLEESGGGLVQAGGSLRLSCAASGRTFNTLSMGWFRQAPGKEREFVAAVSRSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAAATKSNTTAYRLSFDYWGQGTQVTVSS; (SEQ ID NO: 35)QVKLEESGGGLVQAGGSLRLSCAASGRTFSMYRMGWFRQAPGKEREFVGVITRNGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATSGSSYLDAAHVYDYWGQGTQVTVSS; (SEQ ID NO: 36)QVKLEESGGGLVQPGGSLRLSCAASGRTLSSYIVAWFRQAPGKEREFVAGISRRGGNSAYVESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADGSVAGWGRRSVSVSSYDYWGQGTQVTVSS; (SEQ ID NO: 37)QVQLVESGGGLAQAGGSLRLSCAASGRTFSMDPMAWFRQPPGKEREFVAAGSSTGRTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAAPYGANWYRDEYAYWGQGTQVTVSS; (SEQ ID NO: 38)QVQLVESGGGLVQAGGSLRLSCAASIRSFSNRNMGWFRQPPGKEREFVAGISWGGGSTRYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEFGHNIATSSDEYDYWGQGTQVTVSS; (SEQ ID NO: 39)QVKLEESGGGLVQAGGSLRLSCAASERTFSRYPVAWFRQAPGAEREFVAVISSTGTSTYYADSVKGRFTISRDNAKVTVYLQMNNLKREDTAVYFCAVNSQRTRLQDPNEYDYWGQGTQVTVSS; (SEQ ID NO: 45)QVKLEESGGGLVQAGGSLRLSCAASGRTFNTLSMGWFRQAPGKEREFVCAVSRSGGSTYYADSVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVYYCAAAATKSNTTAYRLSFDYWGQGTQVTVSS; (SEQ ID NO: 46)QVKLEESGGGLVQAGGSLRLSCAASGRTFSMYRMGWFRQAPGKEREFVCVITRNGSSTYYADSVKGRFTCSRDNAKNTVYLQMNSLKPEDTALYYCAATSGSSYLDAAHVYDYWGQGTQVTVSS; (SEQ ID NO: 47)QVKLEESGGGLVQPGGSLRLSCAASGRTLSSYIVAWFRQAPGKEREFVCGISRRGGNSAYVESVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVYYCAADGSVAGWGRRSVSVSSYDYWGQGTQVTVSS; (SEQ ID NO: 48)QVQLVESGGGLAQAGGSLRLSCAASGRTFSMDPMAWFRQPPGKEREFVCAGSSTGRTTYYADSVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVYYCAAAPYGANWYRDEYAYWGQGTQVTVSS; (SEQ ID NO: 49)QVQLVESGGGLVQAGGSLRLSCAASIRSFSNRNMGWFRQPPGKEREFVCGISWGGGSTRYADSVKGRFTCSRDNAKKTVYLQMNSLKPEDTAVYYCAAEFGHNIATSSDEYDYWGQGTQVTVSS; and (SEQ ID NO: 50)QVKLEESGGGLVQAGGSLRLSCAASERTFSRYPVAWFRQAPGAEREFVCVISSTGTSTYYADSVKGRFTCSRDNAKVTVYLQMNNLKREDTAVYFCAVNSQRTRLQDPNEYDYWGQGTQVTVSS,

allowing the toxin to bind the isolated or purified single domainantibody or fragment thereof and detecting the bound single domainantibody or fragment thereof using a suitable detection and/or imagingtechnology.